Cd20 binding molecules

ABSTRACT

The present invention relates to CD20 binding molecules and nucleic acid sequences encoding CD20 binding molecules. In particular, the present invention relates to CD20 binding molecules with a high binding affinity, and a low dissociation rate, with regard to human CD20. Preferably, the CD20 binding molecules of the present invention comprise light and/or heavy chain variable regions with fully human frameworks (e.g. human germline frameworks).

The present Application claims priority to U.S. Provisional ApplicationSer. No. 60/471,958 filed May 20, 2003.

FIELD OF THE INVENTION

The present invention relates to CD20 binding molecules and nucleic acidsequences encoding CD20 binding molecules. In particular, the presentinvention relates to CD20 binding molecules with a high bindingaffinity, and a low dissociation rate, with regard to human CD20.Preferably, the CD20 binding molecules of the present invention compriselight and/or heavy chain variable regions with fully human frameworks(e.g. human germline frameworks).

BACKGROUND OF THE INVENTION

The use of antibodies to the CD20 antigen as diagnostic and/ortherapeutic agents for diseases such as B-cell lymphoma has previouslybeen reported. CD20 is a useful marker or target for B-cell lymphomas asthis antigen is expressed at very high densities on the surface ofmalignant B-cells, i.e., B-cells wherein unabated proliferation can leadto B-cell lymphomas.

CD20 (also known as Bp35) is a B-lymphocyte-restricted differentiationantigen that is expressed during early pre-B-cell development andremains until plasma cell differentiation. It is believed by some thatthe CD20 molecule may regulate a step in the B-cell activation processwhich is required for cell cycle initiation and differentiation.Moreover, as noted, CD20 is usually expressed at very high levels onneoplastic (“tumor”) B-cells. The CD20 antigen is appealing for targetedtherapy, because it does not shed, modulate, or internalize.

Previous reported therapies involving anti-CD20 antibodies have involvedthe administration of a therapeutic anti-CD20 antibody either alone orin conjunction with a second radiolabeled anti-CD20 antibody, or achemotherapeutic agent. The Food and Drug Administration has approvedthe therapeutic use of one such anti-CD20 antibody, RITUXAN for use inrelapsed and previously treated low-grade non-Hodgkin's lymphoma (NHL).However, while the use of RITUXAN has generally been reported aseffective for treating B-cell lymphomas, the treated patients are oftensubject to disease relapse.

More recently, RITUXAN was tested for safety, tolerability andpreliminary clinical efficacy for the treatment of 18 patients withSystemic Lupus Erythematosus (SLE) (which are non immunosuppressedpatients). Part of the results of this study were presented in Octoberof 2002 at the American College of Rheumatology (ACR) 66th AnnualScientific Meeting. Of the 18 patients treated, six patients receivedone infusion of RITUXAN at 100 mg/m² (low dose), six patients receivedone infusion of RITUXAN at 375 mg/m² (medium dose), and six patientsreceived four weekly infusions of RITUXAN at 375 mg/m² (high dose).Three of the 12 patients that received the low or medium dose (25%)developed elevated human anti-chimera (HACA) titers at two months, whilethe high dose patients are still being evaluated.

Accordingly, what is needed, are CD20 binding molecules that have a highbinding affinity and low dissociation constant such that treated B-celllymphoma patients do not relapse, and CD20 binding molecules that do notcause, or have a reduced potential to cause, a HACA reaction whenadministered to patients who are not immunosuppressed.

SUMMARY OF THE INVENTION

The present invention provides CD20 binding molecules and nucleic acidsequences encoding CD20 binding molecules. In particular, the presentprovides CD20 binding molecules with a high binding affinity, and a lowdissociation rate, with regard to human CD20. Preferably, the CD20binding molecules of the present invention comprise light and/or heavychain variable regions with fully human frameworks (e.g. human germlineframeworks).

In some embodiments, the present invention provides compositionscomprising a CD20 binding molecule, wherein the CD20 binding moleculecomprises: a) a light chain variable region, or a portion of a lightchain variable region, wherein the light chain variable region (or theportion) comprises; i) a CDRL1 amino acid sequence selected from thegroup consisting of SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5; ii) aCDRL2 amino acid sequence selected from the group consisting of SEQ IDNO:7, SEQ ID NO:9, SEQ ID NO:11, and SEQ ID NO:13; iii) a CDRL3 aminoacid sequence selected from the group consisting of SEQ ID NO:17, SEQ IDNO:19, and SEQ ID NO:21; and b) a heavy chain variable region, or aportion of a heavy chain variable region, wherein the heavy chainvariable region (or the portion) comprises; i) a CDRH1 amino acidsequence selected from the group consisting of SEQ ID NO:23 and SEQ IDNO:25; ii) a CDRH2 amino acid sequence selected from the groupconsisting of SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33,SEQ ID NO:35, SEQ ID NO:37, and SEQ ID NO:39; and iii) a CDRH3 aminoacid sequence selected from the group consisting of SEQ ID NO:43, SEQ IDNO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ IDNO:55, and SEQ ID NO:57.

In other embodiments, the present invention provides compositionscomprising a light chain variable region (or a portion thereof), or anucleic acid sequence (or a portion thereof) encoding a light chainvariable region, wherein the light chain variable region (or the portionthereof) comprises: a) a CDRL1 amino acid sequence selected from thegroup consisting of SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5; b) aCDRL2 amino acid sequence selected from the group consisting of SEQ IDNO:7, SEQ ID NO:9, SEQ ID NO:11, and SEQ ID NO:13; and c) a CDRL3 aminoacid sequence selected from the group consisting of SEQ ID NO:17, SEQ IDNO:19, and SEQ ID NO:21.

In certain embodiments, the present invention provides compositionscomprising a heavy chain variable region (or portion thereof), or anucleic acid sequence (or portion thereof) encoding a heavy chainvariable region, wherein the heavy chain variable region (or portionthereof) comprises: a) a CDRH1 amino acid sequence selected from thegroup consisting of SEQ ID NO:23 and SEQ ID NO:25; b) a CDRH2 amino acidsequence selected from the group consisting of SEQ ID NO:27, SEQ IDNO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, and SEQID NO:39; and c) a CDRH3 amino acid sequence selected from the groupconsisting of SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49,SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, and SEQ ID NO:57.

In additional embodiments, the present invention provides compositionscomprising: a) a light chain variable region, or a first nucleic acidsequence encoding a light chain variable region, wherein the light chainvariable region comprises a CDRL3 amino acid sequence selected from thegroup consisting of SEQ ID NO:17, SEQ ID NO:19, and SEQ ID NO:21; and b)a heavy chain variable region, or a second nucleic acid sequenceencoding a heavy chain variable region, wherein the heavy chain variableregion comprises a CDRH3 amino acid sequence selected from the groupconsisting of SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49,SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, and SEQ ID NO:57.

In some embodiments, the present invention provides compositionscomprising a nucleic acid molecule encoding a light chain variableregion of a CD20 binding molecule, wherein the nucleic acid moleculecomprises; a) a CDRL1 nucleic acid sequence selected from the groupconsisting of SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6; b) a CDRL2nucleic acid sequence selected from the group consisting of SEQ ID NO:8,SEQ ID NO:10, SEQ ID NO:12, and SEQ ID NO:14; and c) a CDRL3 nucleicacid sequence selected from the group consisting of SEQ ID NO:18, SEQ IDNO:20, and SEQ ID NO:22. In other embodiments, the present inventionprovides compositions comprising a nucleic acid molecule encoding aheavy chain variable region of a CD20 binding molecule, wherein thenucleic acid molecule comprises; a) a CDRH1 nucleic acid sequenceselected from the group consisting of SEQ ID NO:24 and SEQ ID NO:26; b)a CDRH2 nucleic acid sequence selected from the group consisting of SEQID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ IDNO:38, and SEQ ID NO:40; and c) a CDRH3 nucleic acid sequence selectedfrom the group consisting of SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48,SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, and SEQ IDNO:58.

In particular embodiments, the present invention provides compositionscomprising a CD20 binding molecule, wherein the CD20 binding moleculecomprises a) a CDRL1 amino acid sequence comprising SEQ ID NO:5; b) aCDRL2 amino acid sequence comprising SEQ ID NO:13; c) a CDRL3 amino acidsequence comprising SEQ ID NO:19; d) a CDRH1 amino acid sequencecomprising SEQ ID NO:25; e) a CDRH2 amino acid sequence comprising SEQID NO:39; and f) a CDRH3 amino acid sequence comprising SEQ ID NO:57. Inother embodiments, the present invention provides compositionscomprising a nucleic acid molecule encoding a light chain variableregion of a CD20 binding molecule, wherein the nucleic acid moleculecomprises a) a CDRL1 nucleic acid sequence comprising SEQ ID NO:6; b) aCDRL2 nucleic acid sequence comprising SEQ ID NO:14; and c) a CDRL3nucleic acid sequence comprising SEQ ID NO:20.

In further embodiments, the present invention provides compositionscomprising a nucleic acid molecule encoding a heavy chain variableregion of a CD20 binding molecule, wherein the nucleic acid moleculecomprises a) a CDRH1 nucleic acid sequence comprising SEQ ID NO:26; b) aCDRH2 nucleic acid sequence comprising SEQ ID NO:40; and c) a CDRH3nucleic acid sequence comprising SEQ ID NO:58. In other embodiments, thepresent invention provides compositions comprising: a) a first nucleicacid sequence encoding a light chain variable region, wherein the firstnucleic acid sequence comprises a CDRL3 nucleic acid sequence selectedfrom the group consisting of SEQ ID NO:18, SEQ ID NO:20, and SEQ IDNO:22; and b) a second nucleic acid sequence encoding a heavy chainvariable region, wherein the second nucleic acid sequence comprises aCDRH3 nucleic acid sequence selected from the group consisting of SEQ IDNO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ IDNO:54, SEQ ID NO:56, and SEQ ID NO:58.

In some embodiments, the present invention provides compositionscomprising: a) a first nucleic acid sequence encoding a light chainvariable region, wherein the light chain variable region comprises; i) aCDRL1 amino acid sequence selected from the group consisting of SEQ IDNO:1, SEQ ID NO:3, and SEQ ID NO:5; ii) a CDRL2 amino acid sequenceselected from the group consisting of SEQ ID NO:7, SEQ ID NO:9, SEQ IDNO:11, and SEQ ID NO:13; and iii) a CDRL3 amino acid sequence selectedfrom the group consisting of SEQ ID NO:17, SEQ ID NO:19, and SEQ IDNO:21; and b) a second nucleic acid sequence encoding a heavy chainvariable region, wherein the heavy chain variable region comprises; i) aCDRH1 amino acid sequence selected from the group consisting of SEQ IDNO:23 and SEQ ID NO:25; ii) a CDRH2 amino acid sequence selected fromthe group consisting of SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ IDNO:33, SEQ ID NO:35, SEQ ID NO:37, and SEQ ID NO:39; and iii) a CDRH3amino acid sequence selected from the group consisting of SEQ ID NO:43,SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53,SEQ ID NO:55, and SEQ ID NO:57.

In some embodiments, the present invention provides a peptide comprisingat least two (or at least three or four) CDRs selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5; SEQ ID NO:7, SEQ IDNO:9, SEQ ID NO:11, SEQ ID NO:13; SEQ ID NO:17, SEQ ID NO:19, and SEQ IDNO:21. In other embodiments, the present invention provides a peptidecomprising at least two (or at least three or four) CDRs selected fromthe group consisting of SEQ ID NO:23, SEQ ID NO:25; SEQ ID NO:27, SEQ IDNO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ IDNO:39; of SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ IDNO:51, SEQ ID NO:53, SEQ ID NO:55, and SEQ ID NO:57.

In certain embodiments, the present invention provides a compositioncomprising a light chain variable region and a heavy chain variableregion, wherein the light chain variable region comprises an amino acidsequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3,SEQ ID NO:5; SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13; SEQID NO:17, SEQ ID NO:19, and SEQ ID NO:21; and wherein the heavy chainvariable region comprises an amino acid sequence selected from SEQ IDNO:23, SEQ ID NO:25; SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ IDNO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39; of SEQ ID NO:43, SEQ IDNO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ IDNO:55, and SEQ ID NO:57.

In certain embodiments, the light chain variable region comprises aportion of a framework (e.g. containing 2 or 3 subregions, such as FR2and FR3). In some embodiments, the light chain variable region comprisesa fully human framework. In other embodiments, the light chain variableregion comprise a human germline framework. In particular embodiments,the light chain variable region comprises an amino acid sequenceselected from SEQ ID NO:59, and 63. In certain embodiments, the heavychain variable region comprises a portion of a framework (e.g.containing 2 or 3 subregions, such as FR2 and FR3). In certainembodiments, the heavy chain variable region comprises a fully humanframework. In other embodiments, the heavy chain variable regioncomprises a human germline framework. In additional embodiments, theheavy chain variable region comprises an amino acid sequence selectedfrom 61, and 65.

In some embodiments, the CD20 binding molecule comprises an antibody orantibody fragment (e.g., Fv, Fab, etc.). In particular embodiments, theCD20 binding molecule comprises the AME 33 Fv or Fab. In otherembodiments, the CD20 binding molecule comprises the AME 5 Fv or Fab. Inadditional embodiments, the CD20 binding molecule comprises an Fv or Fabselected from the group consisting of AME 21E1 Hum, AME 6F1, AME 2C2,AME 1D10, AME 15, AME 18, AME 5-3, AME 1C2, and AME 4H5.

In particular embodiments, the present invention provides fusionconstructs comprising a CD20 binding molecule (e.g. an antibody orantibody fragment) and a fusion partner, such as an enzyme, detectablelabel, carbohydrate molecule, a lipid, etc. In some embodiments, thefusion construct comprises an Fab or Fab′2 that binds human CD20 and anenzyme for converting a pro-drug into an active form.

In certain embodiments, the CD20 binding molecule is contained within ahost cell (e.g. eukaryotic, or prokaryotic host cell). In otherembodiments, the nucleic acid sequence encoding the light and/or heavychain is contained within a plasmid or other expression vector.

In some embodiments, the present invention provides compositionscomprising a CD20 binding molecule that has a binding affinity (K_(d))for human CD20 of 5.0×10⁻¹⁰ M or less (e.g, 5.0×10⁻¹⁰ M−5.0×10⁻¹¹ M). Inother embodiments, the present invention provides compositionscomprising a CD20 binding molecule, wherein the CD20 binding moleculehas a binding affinity (K_(d)) for human CD20 of 5.0×10⁻¹⁰ M or less,and a dissociation rate (koff) for human CD20 of 5.0×10⁻⁴ s⁻¹ or less.

In additional embodiments, the CD20 binding molecule has a bindingaffinity (K_(d)) for human CD20 of 1.5×10⁻¹⁰ M or less. In someembodiments, the CD20 binding molecule has a binding affinity (K_(d))for human CD20 of 1.0×10⁻¹⁰ M or less. In certain embodiments, the CD20binding molecule has a dissociation rate (koff) for human CD20 of2.5×10⁻⁴ s⁻¹ or less. In particular embodiments, the CD20 bindingmolecule has a dissociation rate (koff) for human CD20 of 1.0×10⁻⁴ s⁻¹or less (e.g., 1.0×10⁻⁴ s⁻¹-1.0×10⁻⁵ s⁻¹). In some embodiments, the CD20binding molecule has a dissociation rate (koff) for human CD20 of8.0×10⁻⁵ s⁻¹ or less. In further embodiments, the CD20 binding moleculehas an association rate (kon) for human CD20 of 1.0×10⁵ M⁻¹ s⁻¹ orgreater (e.g., 1.0×10⁵ M⁻¹ s⁻¹−1.0×10⁶ M⁻¹ s⁻¹). In certain embodiments,the CD20 binding molecule has an association rate (kon) for human CD20of 5.0×10⁵ M⁻¹ s⁻¹ or greater.

In some embodiments, the present invention provides methods of treatingB cell lymphoma comprising: a) providing; i) a subject, and ii) acomposition, wherein the composition comprises a CD20 binding moleculeof the present invention; and b) administering the composition to thesubject. In other embodiments, the present invention provides methods oftreating a disease comprising: a) providing; i) a subject with symptomsof the disease, and ii) a composition, wherein the composition comprisesthe CD20 binding molecules of the present invention; and b)administering the composition to the subject such that the symptoms arereduced or eliminated. In particular embodiments, the disease isselected from the group consisting of: relapsed Hodgkin's disease,resistant Hodgkin's disease high grade, low grade and intermediate gradenon-Hodgkin's lymphomas (NHLs), B cell chronic lymphocytic leukemia(B-CLL), lymphoplasmacytoid lymphoma (LPL), mantle cell lymphoma (MCL),follicular lymphoma (FL), diffuse large cell lymphoma (DLCL), Burkitt'slymphoma (BL), AIDS-related lymphomas, monocytic B cell lymphoma,angioimmunoblastic lymphoadenopathy, small lymphocytic; follicular,diffuse large cell; diffuse small cleaved cell; large cell immunoblasticlymphoblastoma; small, non-cleaved; Burkift's and non-Burkitt's;follicular, predominantly large cell; follicular, predominantly smallcleaved cell; and follicular, mixed small cleaved and large celllymphomas.

In some embodiments, the present invention provides a method of treatinga disease (e.g. cancer) in an animal requiring such treatment, whichmethod comprises administering the animal an effective amount of theCD20 binding molecules of the present invention. In certain embodiments,the present invention provides the CD20 binding molecules of the presentinvention for use as a medicament. In other embodiments, the presentinvention provides the CD20 binding molecules of the present inventionin the manufacture of a medicament for the treatment of disease (e.g.NHL). In particular embodiments, the present invention provides apharmaceutical for the treatment of a disease (e.g. cancer)characterized in that it contain the CD20 binding molecules of thepresent invention as an active substance.

In preferred embodiments, the subject (patient) is non-immunosuppressed.For example, the patient may have a disease such as systemic lupuserythematosus (SLE). Preferably, once the non-immunosuppressed subjectis administered the CD20 binding molecules, no (or negligible) HACAresponse (human anti-chimeric antibody response) is generated. Incertain embodiments, the dosage administered to the subject is about 375mg/m per week for four weeks (without generating a HACA response). Insome embodiments, the dosage administered is about 50-300 mg/m² perweek, or about 100-200 mg/m² per week, for four weeks (e.g. for treatingChronic Lymphocytic Leukemia (CLL)).

In some embodiments, the present invention provides methods of treatingB cell lymphoma comprising: a) providing; i) a subject, and ii) acomposition, wherein the composition comprises CD20 binding moleculesthat have a binding affinity (K_(d)) for human CD20 of 5.0×10⁻¹⁰ M orless, and a dissociation rate (koff) for human CD20 of 5.0×10⁻⁴ s⁻¹ orless; and b) administering the composition to the subject.

In certain embodiments, the present invention provides methods fordepleting peripheral B cells in a subject in need of such treatmentcomprising: a) providing; i) a subject, and ii) a composition, whereinthe composition comprises CD20 binding molecules that have a bindingaffinity (K_(d)) for human CD20 of 5.0×10⁻¹⁰ M or less, and adissociation rate (koff) for human CD20 of 5.0×10⁻⁴ s⁻¹ or less; and b)administering the composition to the subject.

In some embodiments, the light chain variable region comprises a fullyhuman framework. In other embodiments, the light chain variable regioncomprise a human germline framework. In particular embodiments, thelight chain variable region comprises an amino acid sequence selectedfrom SEQ ID NO:59 and 63. In certain embodiments, the heavy chainvariable region comprises a fully human framework. In other embodiments,the heavy chain variable region comprises a human germline framework. Inadditional embodiments, the heavy chain variable region comprises anamino acid sequence selected from 61 and 65.

In some embodiments, the CD20 binding molecule comprises an antibody orantibody fragment (e.g., Fv, Fab, etc.). In particular embodiments, theCD20 binding molecule comprises the AME 33 Fv or Fab. In otherembodiments, the CD20 binding molecule comprises the AME 5 Fv or Fab. Inadditional embodiments, the CD20 binding molecule comprises an Fv or Fabselected from the group consisting of AME 21E1 Hum, AME 6F1, AME 2C2,AME 1D10, AME 15, AME 18, AME 5-3, AME 1C2, AME 4H5.

In other embodiments, the CD20 binding molecule mediatesantibody-dependent cell mediated cytotoxicity (ADCC) at approximatelythe same EC50 level as the C2B8 antibody. In further embodiments, theCD20 binding molecule mediates antibody-dependent cell mediatedcytotoxicity (ADCC), with human peripheral blood mononuclear cells aseffectors and B lymphoma cells as targets, at approximately the sameEC50 level as the C2B8 antibody. In some embodiments, the CD20 bindingmolecule mediates antibody-dependent cell mediated cytotoxicity (ADCC)at approximately 1.5 to 2.0 times that of the EC50 level as the C2B8antibody (the C2B8 antibody is deposited with the ATCC as number 69119).In additional embodiments, the CD20 binding molecule mediatesantibody-dependent cell mediated cytotoxicity (ADCC), with humanperipheral blood mononuclear cells as effectors and B lymphoma cells astargets, at approximately 1.5 to 2.0 times the EC50 level as the C2B8antibody. In further embodiments, the CD20 binding molecule mediatesantibody-dependent cell mediated cytotoxicity (ADCC) at an EC50 level 10times that of the C2B8 antibody or greater. In additional embodiments,the CD20 binding molecule mediates antibody-dependent cell mediatedcytotoxicity (ADCC), with human peripheral blood mononuclear cells aseffectors and B lymphoma cells as targets, at an EC50 level 10 timesthat of the C2B8 antibody or greater.

In some embodiments, the CD20 binding molecules comprise a humangermline light chain framework. In certain embodiments, this light chainhuman germline framework is selected from V1-11, V1-13, V1-16, V1-17,V1-18, V1-19, V1-2, V1-20, V1-22, V1-3, V1-4, V1-5, V1-7, V1-9, V2-1,V2-11, V2-13, V2-14, V2-15, V2-17, V2-19, V2-6, V2-7, V2-8, V3-2, V3-3,V3-4, V4-1, V4-2, V4-3, V4-4, V4-6, V5-1, V5-2, V5-4, and V5-6.

In other embodiments, the CD20 binding molecules comprise a humangermline heavy chain framework. In particular embodiments, this heavychain human germline framework is selected from VH1-18, VH1-2, VH1-24,VH1-3, VH1-45, VH1-46, VH1-58, VH1-69, VH1-8, VH2-26, VH2-5, VH2-70,VH3-11, VH3-13, VH3-15, VH3-16, VH3-20, VH3-21, VH3-23, VH3-30, VH3-33,VH3-35, VH3-38, VH3-43, VH3-48, VH3-49, VH3-53, VH3-64, VH3-66, VH3-7,VH3-72, VH3-73, VH3-74, VH3-9, VH4-28, VH4-31, VH4-34, VH4-39, VH4-4,VH4-59, VH4-61, VH5-51, VH6-1, and VH7-81.

In some embodiments, the CD20 binding molecule is a CD20 binding peptideor polypeptide. In certain embodiments, the CD20 binding peptidecomprises an anti-CD20 antibody or anti-CD20 antibody fragment (e.g.,Fv, Fab, F(ab′)₂, etc). In other embodiments, the peptide comprises alight and/or heavy chain variable region. In particular embodiments, thelight chain variable region and/or heavy chain variable region comprisesa framework region or at least a portion of a framework region (e.g.containing 2 or 3 subregions, such as FR2 and FR3). In certainembodiments, at least FRL1, FRL2, FRL3, or FRL4 is fully human. In otherembodiments, at least FRH1, FRH2, FRH3, or FRH4 is fully human. In someembodiments, at least FRL1, FRL2, FRL3, or FRL4 is a germline sequence(e.g. human germline). In other embodiments, at least FRH1, FRH2, FRH3,or FRH4 is a germline sequence (e.g. human germline). In preferredembodiments, the framework region is a fully human framework region(e.g. the human framework regions shown in FIGS. 4-5 and 8-9). In someembodiments, the framework region comprises SEQ ID NO: 71, 72, 73, 74,79, 80, 81, 82, or combinations thereof. In other embodiments, theframework region comprises SEQ ID NO: 87, 88, 89, 90, 95, 96, 97, 98, orcombinations thereof.

In some embodiments, the present invention provides a computer readablemedium that encodes a representation of a nucleic acid or amino acidsequence selected from SEQ ID NOs: 1-70, or the complement thereof. Incertain embodiments, the representation of these sequences, whendelivered to a computer processor, may be displayed to a user (e.g.,over the internet).

In certain embodiments, the present invention provides nucleic acidsequences that hybridize (under low, medium or high stringencyconditions) to the nucleic acid sequences found in SEQ ID NOs:1-70, orthe nucleic acid sequences encoding the amino acid sequences found inSEQ ID NOs:1-70.

In some embodiments, the present invention provides the complement ofthe nucleic acid sequences described herein (see e.g. Tables 1-2, andFIGS. 2, 3, 6, 7, 10, and 11). In some embodiments, the presentinvention provides sequences that hybridize under high, medium or lowstringency with the nucleic acid sequences described herein (see e.g.Tables 1-2, and FIGS. 2, 3, 6, 7, 10, and 11).

In certain embodiments, the affinity constant (Kd) and association rate(Kon) are determined in a IgG cell binding assay (i.e. cells expressinghuman CD20 on their surface), using KinExa equilibrium software (e.g.from Sapidyne Instruments, Boise, Id.). In some embodiments, theaffinity constant and association rate constant are determined by akinetic exclusion assay (See, e.g., Chiu et al., (2001) Anal. Chem.,73:5477-5484; Blake, et al., (1996) Journal of Biological Chemistry,271:27677-27685; Hongo, et al., (2000) Hybridoma, 19:303-315;Khosraviani, et al., (2000) Bioconjugate Chemistry, 11:267-277; andPowers, et al., (2001) Journal of Immunological Methods, 251:123-135,all of which are herein incorporated by reference). In particularembodiments, the kinetic exclusion assay is performed with a KinExAinstrument (e.g., KinExA™3000 from Sapidyne Instruments, Boise, Id.), orsimilar device.

In other embodiments, the CD20 binding molecule comprises a Fab, andfurther comprises one or more constant regions (e.g., CH2 and/or CH3,see FIGS. 10-11). In particular embodiments, the CD20 binding moleculecomprises an antibody (e.g., an antibody comprising a fully humanframework with synthetic CDR sequences). In certain embodiments, theantibody comprises an altered (e.g., mutated) Fc region. For example, insome embodiments, the Fc region has been altered to reduce or enhancethe effector functions of the antibody. In some embodiments, the Fcregion is an isotype selected from IgM, IgA, IgG, IgE, or other isotype.

In some embodiments, amino acid modification(s) are introduced into theCH2 domain of an Fc region of a CD20 binding molecule. Useful amino acidpositions for modification in order to generate a variant IgG Fc regionwith altered Fc gamma receptor (FcγR) binding affinity or activityinclude any one or more of the following amino acid positions: 268, 269,270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295,296, 298, 300 301, 303, 305, 307, 309, 331, 333, 334, 335, 337, 338,340, 360, 373, 376, 416, 419, 430, 434, 435, 437, 438 or 439 of the Fcregion of a CD20 binding molecule. In preferred embodiments, the parentFc region used as the template to generate such variants comprises ahuman IgG Fc region. In some embodiments, to generate an Fc regionvariant with reduced binding to the FcγR one may introduce an amino acidmodification at any one or more of the following amino acid positions:252, 254, 265, 268, 269, 270, 272, 278, 289, 292, 293, 294, 295, 296,298, 300, 301, 303, 322, 324, 327, 329, 333, 335, 338, 340, 373, 376,382, 388, 389, 414, 416, 419, 434, 435, 437, 438 or 439 of the Fc regionof a CD20 binding molecule. In particular embodiments, Fc regionvariants with improved binding to one or more FcγRs may also be made.Such Fc region variants may comprise an amino acid modification at anyone or more of the following amino acid positions: 280, 283, 285, 286,290, 294, 295, 298, 300, 301, 305, 307, 309, 312, 315, 331, 333, 3.34,337, 340, 360, 378, 398 or 430 of the Fc region of a CD20 bindingmolecule. In certain embodiments, the amino acid modification is Y300I.

In other embodiments, the amino acid modification is Y300L. In someembodiments, the amino acid modification is Q295K or Q295L. In certainembodiments, the amino acid modification is E294N. In other embodiments,the amino acid modification at position 296 is Y296P. In someembodiments, the amino acid modification at position 298 is S298P. Inother embodiments, the amino acid modification is S298N, S298P, S298V orS298D.

In certain embodiments, the CD20 binding molecule comprises a heavychain constant region mutation. In other embodiments, the CD20 bindingmolecule comprises a heavy chain constant region with a mutationselected from D280H and K290S.

Alternatively or additionally, it may be useful to combine amino acidmodifications with one or more further amino acid modifications thatalter C1q binding and/or the complement dependent cytotoxicity functionof the Fc region of a CD20 binding molecule. The starting polypeptide ofparticular interest may be one that binds to C1q and displays complementdependent cytotoxicity (CDC). Amino acid substitutions described hereinmay serve to alter the ability of the starting polypeptide to bind toC1q and/or modify its complement dependent cytotoxicity function (e.g.,to reduce and preferably abolish these effector functions). However,polypeptides comprising substitutions at one or more of the describedpositions with improved C1q binding and/or complement dependentcytotoxicity (CDC) function are contemplated herein. For example, thestarting polypeptide may be unable to bind C1q and/or mediate CDC andmay be modified according to the teachings herein such that it acquiresthese further effector functions. Moreover, polypeptides withpre-existing C1q binding activity, optionally further having the abilityto mediate CDC may be modified such that one or both of these activitiesare enhanced. Amino acid modifications that alter C1q and/or modify itscomplement dependent cytotoxicity function are described, for example,in WO0042072, which is hereby incorporated by reference.

As disclosed above, one can design an Fc region of a CD20 bindingmolecule with altered effector function, e.g., by modifying C1q bindingand/or FcγR binding and thereby changing CDC activity and/or ADCCactivity. For example, one can generate a variant Fc region of a CD20binding molecule with improved C1q binding and improved FcγRIIIbinding(e.g., having both improved ADCC activity and improved CDC activity).Alternatively, where one desires that effector function be reduced orablated, one may engineer a variant Fc region with reduced CDC activityand/or reduced ADCC activity. In other embodiments, one may increaseonly one of these activities, and optionally also reduce the otheractivity (e.g., to generate an Fc region variant with improved ADCCactivity, but reduced CDC activity and vice versa).

Fc mutations can also be introduced in the CD20 binding molecules of thepresent invention to alter their interaction with the neonatal Fcreceptor (FcRn) and improve their pharmacokinetic properties. Severalexperiments suggest that the interaction between the Fc region of anantibody and the FcRn plays a role in the persistence of immunoglobulinsin serum. For instance, an unusually short serum half-life is observedfor IgG molecules in mice that lack a functional FcRn. Fc mutations thatimprove binding to the FcRn appear to prolong serum half-life and,conversely, mutations in the rat FcRn that result in tighter IgG bindingalso improve serum half-life. A collection of human Fc variants withimproved binding to the FcRn has also been described (Shields et al.,(2001) High resolution mapping of the binding site on human IgG1 forFcγRI, FcγRII, FcγRIII, and FcRn and design of IgG1 variants withimproved binding to the FcγR, J. Biol. Chem. 276:6591-6604). It has beenreported that the increased binding affinity of IgG molecules for theFcRn observed at low pH (e.g., during pinocytosis or fluid phaseendocytosis of IgG molecules from serum) impacts serum half-life (Ghetieet al., (1997) Increasing the serum persistence of an IgG fragment byrandom mutagenesis, Nat. Biotechnol. 15:637-640; Medesan et al., (1998)Comparative studies of rat IgG to further delineate the Fc:FcRninteraction site. Eur. J. Immunol. 28:2092-2100; Kim et al., (1999)Mapping the site on human IgG for binding of the MHC class I-relatedreceptor, FcRn, Eur. J. Immunol. 29:2819-2825; Acqua et al., (2002)Increasing the affinity of a human IgG1 for the neonatal Fc receptor:biological consequences, J. Immunol. 169:5171-5180). However, mutationsthat increase binding at high pH appear to adversely affect serumhalf-life (Acqua et al., (2002) Increasing the affinity of a human IgG1for the neonatal Fc receptor: biological consequences, J. Immunol.169:5171-5180). All of the articles above are herein incorporated byreference. Therefore, Fc mutations could be introduced in the CD20binding molecules of the present invention in order to increase theiraffinity for the FcRn at low pH but maintain or decrease their affinityfor the FcRn at higher pH.

Another type of amino acid substitution serves to alter theglycosylation pattern of the Fc region of a CD20 binding molecule. Thismay be achieved, for example, by deleting one or more glycosylationsite(s) found in the polypeptide, and/or adding one or moreglycosylation sites that are not present in the polypeptide.Glycosylation of an Fc region is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The peptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these peptide sequences in a polypeptidecreates a potential glycosylation site. O-linked glycosylation refers tothe attachment of one of the sugars N-aceylgalactosamine, galactose, orxylose to a hydroxyamino acid, most commonly serine or threonine,although 5-hydroxyproline or 5-hydroxylysine may also be used.

Addition of glycosylation sites to the Fc region of a CD20 bindingmolecule is conveniently accomplished by altering the amino acidsequence such that it contains one or more of the above-describedtripeptide sequences (for N-linked glycosylation sites). An exemplaryglycosylation variant has an amino acid substitution of residue Asn 297of the heavy chain. The alteration may also be made by the addition of,or substitution by, one or more serine or threonine residues to thesequence of the original polypeptide (for O-linked glycosylation sites).

In certain embodiments, the CD20 binding molecules of the presentinvention are expressed in cells that expressbeta(1,4)-N-acetylglucosaminyltransferase III (GnT III), such that GnTIII adds GlcNAc to the CD20 binding molecules. Methods for producingbinding molecules in such a fashion are provided in WO9954342,WO03011878, patent publication 20030003097A1, and Umana et al., NatureBiotechnology, 17:176-180, February 1999; all of which are hereinspecifically incorporated by reference in their entireties.

In certain embodiments, the present invention provides kits comprising:a) a CD20 binding molecule of the present invention; and b) instructionsfor using the CD20 binding molecule to treat a disease in a subject orinstructions for employing the CD20 binding molecule for scientificresearch or diagnostic purposes (e.g., for performing ELISA assays,etc.). In some embodiments, the present invention provides cell linesstably or transiently transfected with nucleic acid sequences encodingthe CD20 binding molecules of the present invention.

DESCRIPTION OF THE FIGURES

FIG. 1 shows a schematic representation of an IgG molecule with thevarious regions and sections labeled. The CDRs and framework regions(FR) of one of the two variable region light chains, and one of the twovariable region heavy chains, are also labeled.

FIG. 2A shows the amino acid sequence of the light chain variable regionof AME 33 (SEQ ID NO:59), and FIG. 2B shows the nucleic acid sequence ofthe light chain variable region of AME 33 (SEQ ID NO:60).

FIG. 3A shows the amino acid sequence of the heavy chain variable regionof AME 33 (SEQ ID NO:61), and FIG. 3B shows the nucleic acid sequence ofthe heavy chain variable region of AME 33 (SEQ ID NO:62).

FIG. 4A shows the amino acid sequence of the fully human light chainframework region VkIII (A27) (DPK22) with interspersed CDRs. The fourframework sub-regions are labeled as follows: FRL1 (SEQ ID NO:71), FRL2(SEQ ID NO:72), FRL3 (SEQ ID NO:73), and FRL4 (SEQ ID NO:74). FIG. 4Bshows the nucleic acid sequence of the human light chain frameworkregion VkIII (A27) (DPK22) with interspersed CDRs. The four frameworksub-regions are labeled as follows: FRL1 (SEQ ID NO:75), FRL2 (SEQ IDNO:76), FRL3 (SEQ ID NO:77), and FRL4 (SEQ ID NO:78).

FIG. 5A shows the amino acid sequence of the human heavy chain frameworkregion VH5-51 (DP-73) with interspersed CDRs. The four frameworksub-regions are labeled as follows: FRH1 (SEQ ID NO:79), FRH2 (SEQ IDNO:80), FRH3 (SEQ ID NO:81), and FRH4 (SEQ ID NO:82). FIG. 5B shows thenucleic acid sequence of the human heavy chain framework region V5-51(DP-73) with interspersed CDRs. The four framework sub-regions arelabeled as follows: FRH1 (SEQ ID NO:83), FRH2 (SEQ ID NO:84), FRH3 (SEQID NO:85), and FRH4 (SEQ ID NO:86).

FIG. 6A shows the amino acid sequence of the light chain variable regionof AME 5 (SEQ ID NO:63), and FIG. 6B shows the nucleic acid sequence ofthe light chain variable region of AME 5 (SEQ ID NO:64).

FIG. 7A shows the amino acid sequence of the heavy chain variable regionof AME 5 (SEQ ID NO:65), and FIG. 7B shows the nucleic acid sequence ofthe heavy chain variable region of AME 5 (SEQ ID NO:66).

FIG. 8A shows the amino acid sequence of the human light chain frameworkregion VkI (DPK4) (A20) with interspersed CDRs. The four frameworksub-regions are labeled as follows: FRL1 (SEQ ID NO:87), FRL2 (SEQ IDNO:88), FRL3 (SEQ ID NO:89), and FRL4 (SEQ ID NO:90). FIG. 8B shows thenucleic acid sequence of the human light chain framework region VkI(DPK4) (A20) with interspersed CDRs. The four framework sub-regions arelabeled as follows: FRL1 (SEQ ID NO:91), FRL2 (SEQ ID NO:92), FRL3 (SEQID NO:93), and FRL4 (SEQ ID NO:94).

FIG. 9A shows the amino acid sequence of the human heavy chain frameworkregion VHI DP7/21-2 with interspersed CDRs. The four frameworksub-regions are labeled as follows: FRH1 (SEQ ID NO:95), FRH2 (SEQ IDNO:96), FRH3 (SEQ ID NO:97), and FRH4 (SEQ ID NO:98). FIG. 9B shows thenucleic acid sequence of the human heavy chain framework region VHIDPI7/21-2 with interspersed CDRs. The four framework sub-regions arelabeled as follows: FRH1 (SEQ ID NO:99), FRH2 (SEQ ID NO:100), FRH3 (SEQID NO:101), and FRH4 (SEQ ID NO:102).

FIG. 10A shows the complete amino acid sequence of the light chain ofAME 33 (SEQ ID NO:67), and FIG. 10B shows the complete nucleic acidsequence of the light chain of AME 33 (SEQ ID NO:68).

FIG. 11A shows the complete amino acid sequence of the heavy chain ofAME 33 (SEQ ID NO:69), and FIG. 11B shows the complete nucleic acidsequence of the heavy chain of AME 33 (SEQ ID NO:70).

FIGS. 12 and 13 show the results of the ELISA binding assay described inExample 2.

FIG. 14 shows the results of the live B lymphoma Fab binding assaysdescribed in Example 3.

FIG. 15 shows the results of the ADCC assay described in Example 5.

FIG. 16 shows the ADCC activity of the glycoengineered CD20 bindingmolecule described in Example 6.

FIG. 17 shows the results of an in vivo tumor inhibition assay describedin Example 9 involving the AME 33 antibody and the C2B8 antibody.

DEFINITIONS

To facilitate an understanding of the invention, a number of terms aredefined below.

The term “antibody,” as used herein, is intended to refer toimmunoglobulin molecules comprised of four polypeptide chains, two heavy(H) chains and two light (L) chains inter-connected by disulfide bonds.Each heavy chain is comprised of a heavy chain variable region(abbreviated herein as HCVR or VH) and a heavy chain constant region.The heavy chain constant region is comprised of three domains, CH1, CH2and CH3 (see FIG. 1). Each light chain is comprised of a light chainvariable region (abbreviated herein as LCVR or VL) and a light chainconstant region. The light chain constant region is comprised of onedomain, CL (see FIG. 1). The VH and VL regions can be further subdividedinto regions of hypervariability, termed complementarity determiningregions (CDRs), interspersed with regions that are more conserved,termed framework regions (FR). Each variable region (VH or VL) contains3 CDRs, designated CDR1, CDR2 and CDR3 (see FIGS. 1, 4, and 5). Eachvariable region also contains 4 framework sub-regions, designated FR1,FR2, FR3 and FR4 (see FIGS. 1, 4, and 5).

As used herein, the term “antibody fragments” refers to a portion of anintact antibody. Examples of antibody fragments include, but are notlimited to, linear antibodies, single-chain antibody molecules, Fv, Faband F(ab′)₂ fragments, and multispecific antibodies formed from antibodyfragments. The antibody fragments preferably retain at least part of theheavy and/or light chain variable region.

As used herein, the terms “complementarity determining region” and “CDR”refer to the regions that are primarily responsible for antigen-binding.There are three CDRs in a light chain variable region (CDRL1, CDRL2, andCDRL3), and three CDRs in a heavy chain variable region (CDRH1, CDRH2,and CDRH3). The residues that make up these six CDRs have beencharacterized by Kabat and Chothia as follows: residues 24-34 (CDRL1),50-56 (CDRL2) and 89-97 (CDRL3) in the light chain variable region and31-35 (CDRH1), 50-65 (CDRH2) and 95-102 (CDRH3) in the heavy chainvariable region; Kabat et al., (1991) Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md., herein incorporated by reference;and residues 26-32 (CDRL1), 50-52 (CDRL2) and 91-96 (CDRL3) in the lightchain variable region and 26-32 (CDRH1), 53-55 (CDRH2) and 96-101(CDRH3) in the heavy chain variable region; Chothia and Lesk (1987) J.Mol. Biol. 196: 901-917, herein incorporated by reference. Unlessotherwise specified, the terms “complementarity determining region” and“CDR” as used herein, include the residues that encompass both the Kabatand Chothia definitions (i.e., residues 24-34 (CDRL1), 50-56 (CDRL2),and 89-97 (CDRL3) in the light chain variable region; and 26-35 (CDRH1),50-65 (CDRH2), and 95-102 (CDRH3)). Also, unless specified, as usedherein, the numbering of CDR residues is according to Kabat.

As used herein, the term “framework” refers to the residues of thevariable region other than the CDR residues as defined herein. There arefour separate framework sub-regions that make up the framework: FR1,FR2, FR3, and FR4 (See FIGS. 1, 4, and 6). In order to indicate if theframework sub-region is in the light or heavy chain variable region, an“L” or “H” may be added to the sub-region abbreviation (e.g., “FRL1”indicates framework sub-region 1 of the light chain variable region).Unless specified, the numbering of framework residues is according toKabat. It is noted that, in certain embodiments, the CD20 bindingmolecules of the present invention may have less than a completeframework (e.g. the CD20 binding molecule may have a portion of aframework that only contains one or more of the four sub-regions).

As used herein, the term “fully human framework” means a framework withan amino acid sequence found naturally in humans. Examples of fullyhuman frameworks, include, but are not limited to, KOL, NEWM, REI, EU,TUR, TEI, LAY and POM (See, e.g., Kabat et al., (1991) Sequences ofProteins of Immunological Interest, US Department of Health and HumanServices, NIH, USA; and Wu et al., (1970) J. Exp. Med. 132, 211-250,both of which are herein incorporated by reference).

As used herein, the terms “subject” and “patient” refer to any animal,such as a mammal like a dog, cat, bird, livestock, and preferably ahuman.

As used herein, the term “codon” or “triplet” refers to a group of threeadjacent nucleotide monomers which specify one of the naturallyoccurring amino acids found in polypeptides. The term also includescodons which do not specify any amino acid. It is also noted that, dueto the degeneracy of the genetic code, there are many codons that codefor the same amino acid. As such, many of the bases of the nucleic acidsequences of the present invention (see, e.g., Tables 1 and 2) can bechanged without changing the actual amino acid sequence that is encoded.The present invention is intended to encompass all such nucleic acidsequences.

As used herein, the terms “an oligonucleotide having a nucleotidesequence encoding a polypeptide,” “polynucleotide having a nucleotidesequence encoding a polypeptide,” and “nucleic acid sequence encoding apeptide” means a nucleic acid sequence comprising the coding region of aparticular polypeptide. The coding region may be present in a cDNA,genomic DNA, or RNA form. When present in a DNA form, theoligonucleotide or polynucleotide may be single-stranded (i.e., thesense strand) or double-stranded. Suitable control elements such asenhancers/promoters, splice junctions, polyadenylation signals, etc. maybe placed in close proximity to the coding region of the gene if neededto permit proper initiation of transcription and/or correct processingof the primary RNA transcript. Alternatively, the coding region utilizedin the expression vectors of the present invention may containendogenous enhancers/promoters, splice junctions, intervening sequences,polyadenylation signals, etc., or a combination of both endogenous andexogenous control elements.

Also, as used herein, there is no size limit or size distinction betweenthe terms “oligonucleotide” and “polynucleotide.” Both terms simplyrefer to molecules composed of nucleotides. Likewise, there is no sizedistinction between the terms “peptide” and “polypeptide.” Both termssimply refer to molecules composed of amino acid residues.

As used herein, the terms “complementary” or “complementarity” are usedin reference to polynucleotides (i.e., a sequence of nucleotides)related by the base-pairing rules. For example, the sequence “5′-A-G-T3”, is complementary to the sequence “3-T-C-A-5′”. Complementarity maybe “partial”, in which only some of the nucleic acids' bases are matchedaccording to the base pairing rules, or, there may be “complete” or“total” complementarity between the nucleic acids. The degree ofcomplementarity between nucleic acid strands has significant effects onthe efficiency and strength of hybridization.

As used herein, the term “the complement of” a given sequence is used inreference to the sequence that is completely complementary to thesequence over its entire length. For example, the sequence 5′-A-G-T-A-3′is “the complement” of the sequence 3′-T-C-A-T-5′. The present inventionalso provides the complement of the sequences described herein (e.g.,the complement of the nucleic acid sequences in SEQ ID NOs:1-70).

As used herein, the term “hybridization” is used in reference to thepairing of complementary nucleic acids. Hybridization and the strengthof hybridization (i.e., the strength of the association between thenucleic acids) is impacted by such factors as the degree ofcomplementarity between the nucleic acids, stringency of the conditionsinvolved, the T_(m) of the formed hybrid, and the G:C ratio within thenucleic acids.

As used herein the term “stringency” is used in reference to theconditions of temperature, ionic strength, and the presence of othercompounds such as organic solvents, under which nucleic acidhybridizations are conducted. Those skilled in the art will recognizethat “stringency” conditions may be altered by varying the parametersjust described either individually or in concert. With “high stringency”conditions, nucleic acid base pairing will occur only between nucleicacid fragments that have a high frequency of complementary basesequences (e.g., hybridization under “high stringency” conditions mayoccur between homologs with about 85-100% identity, preferably about70-100% identity). With medium stringency conditions, nucleic acid basepairing will occur between nucleic acids with an intermediate frequencyof complementary base sequences (e.g., hybridization under “mediumstringency” conditions may occur between homologs with about 50-70%identity). Thus, conditions of “weak” or “low” stringency are oftenrequired with nucleic acids that are derived from organisms that aregenetically diverse, as the frequency of complementary sequences isusually less.

“High stringency conditions” when used in reference to nucleic acidhybridization comprise conditions equivalent to binding or hybridizationat 42 C in a solution consisting of 5×SSPE (43.8 g/l NaCl, 6.9 g/lNaH₂PO₄H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,5× Denhardt's reagent [50× Denhardt's contains per 500 ml: 5 g Ficoll(Type 400, Pharmacia), 5 g BSA (Fraction V; Sigma)] and 100 μg/mldenatured salmon sperm DNA, followed by washing in a solution comprising0.1×SSPE, 1.0% SDS at 42 C when a probe of about 500 nucleotides inlength is employed.

“Medium stringency conditions” when used in reference to nucleic acidhybridization comprise conditions equivalent to binding or hybridizationat 42° C. in a solution consisting of 5×SSPE, 0.5% SDS, 5× Denhardt'sreagent and 100 μg/ml denatured salmon sperm DNA, followed by washing ina solution comprising 1.0×SSPE, 1.0% SDS at 42 C when a probe of about500 nucleotides in length is employed.

“Low stringency conditions” comprise conditions equivalent to binding orhybridization at 42 C in a solution consisting of 5×SSPE, 0.1% SDS, 5×Denhardt's reagent and 100 g/ml denatured salmon sperm DNA, followed bywashing in a solution comprising 5×SSPE, 0.1% SDS at 42 C when a probeof about 500 nucleotides in length is employed.

The term “isolated” when used in relation to a nucleic acid, as in “anisolated oligonucleotide” or “isolated polynucleotide” or “isolatednucleic acid sequence encoding a CD20 binding molecule” (see, e.g.,Tables 1-2) refers to a nucleic acid sequence that is identified andseparated from at least one contaminant nucleic acid with which it isordinarily associated (e.g. host cell proteins).

As used herein, the terms “portion” when used in reference to anucleotide sequence (as in “a portion of a given nucleotide sequence”)refers to fragments of that sequence. The fragments may range in sizefrom ten nucleotides to the entire nucleotide sequence minus onenucleotide (e.g., 10 nucleotides, 20, 30, 40, 50, 100, 200, etc.).

As used herein, the term “portion” when in reference to an amino acidsequence (as in “a portion of a given amino acid sequence”) refers tofragments of that sequence. The fragments may range in size from sixamino acids to the entire amino acid sequence minus one amino acid(e.g., 6 amino acids, 10, 20, 30, 40, 75, 200, etc.).

As used herein, the term “purified” or “to purify” refers to the removalof contaminants from a sample. For example, CD20 specific antibodies maybe purified by removal of contaminating non-immunoglobulin proteins;they are also purified by the removal of immunoglobulins that do notbind to the same antigen. The removal of non-immunoglobulin proteinsand/or the removal of immunoglobulins that do not bind the particularantigen results in an increase in the percentage of antigen specificimmunoglobulins in the sample. In another example, recombinantantigen-specific polypeptides are expressed in bacterial host cells andthe polypeptides are purified by the removal of host cell proteins; thepercentage of recombinant antigen-specific polypeptides is therebyincreased in the sample.

As used herein, the term “vector” is used in reference to nucleic acidmolecules that transfer DNA segment(s) from one cell to another. Theterm “vehicle” is sometimes used interchangeably with “vector.”

The term “expression vector” as used herein refers to a recombinant DNAmolecule containing a desired coding sequence and appropriate nucleicacid sequences necessary for the expression of the operably linkedcoding sequence in a particular host organism. Nucleic acid sequencesnecessary for expression in prokaryotes usually include a promoter, anoperator (optional), and a ribosome binding site, often along with othersequences. Eukaryotic cells are known to utilize promoters, enhancers,and termination and polyadenylation signals.

As used herein, the term “host cell” refers to any eukaryotic orprokaryotic cell (e.g., bacterial cells such as E. Coli, yeast cells,mammalian cells such as PER.C6™ (Crucell, The Netherlands) and CHOcells, avian cells, amphibian cells, plant cells, fish cells, and insectcells), whether located in vitro or in vivo. For example, host cells maybe located in a transgenic animal.

As used herein, the terms “computer memory” and “computer memory device”refer to any storage media readable by a computer processor. Examples ofcomputer memory include, but are not limited to, RAM, ROM, computerchips, digital video discs (DVDs), compact discs (CDs), hard disk drives(HDD), and magnetic tapes.

As used herein, the term “computer readable medium” refers to any deviceor system for storing and providing information (e.g., data andinstructions) to a computer processor. Examples of computer readablemedia include, but are not limited to, DVDs, CDs, hard disk drives,magnetic tapes and servers for streaming media over networks.

As used herein, the phrase “computer readable medium encodes arepresentation” of a nucleic acid or amino acid sequence, refers tocomputer readable medium that has stored thereon information, that whendelivered to a processor, allows the nucleic or amino acid sequence tobe displayed to a user (e.g., printed out or presented on a displayscreen).

As used herein, the terms “processor” and “central processing unit” or“CPU” are used interchangeably and refer to a device that is able toread a program from a computer memory (e.g., ROM or other computermemory) and perform a set of steps according to the program.

As used herein, the term “Fc region” refers to a C-terminal region of animmunoglobulin heavy chain (e.g., as shown in FIG. 1). The “Fc region”may be a native sequence Fc region or a variant Fc region (e.g., withincreased or decreased effector functions).

As used herein, an Fc region may possess “effector functions” that areresponsible for activating or diminishing a biological activity (e.g.,in a subject). Examples of effector functions include, but are notlimited to: C1 q binding; complement dependent cytotoxicity (CDC); Fcreceptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC);phagocytosis; down regulation of cell surface receptors (e.g., B cellreceptor; BCR), etc. Such effector functions may require the Fc regionto be combined with a binding domain (e.g., an antibody variable domain)and can be assessed using various assays (e.g. Fc binding assays, ADCCassays, CDC assays, etc.).

As used herein, an “isolated” peptide, polypeptide, or protein is onethat has been identified and separated and/or recovered from a componentof its natural environment. Contaminant components of its naturalenvironment are materials that would interfere with diagnostic ortherapeutic uses for the polypeptide, and may include enzymes, hormones,and other proteinaceous or non-proteinaceous solutes. In certainembodiments, the isolated polypeptide is purified (1) to greater than95% by weight of polypeptides as determined by the Lowry method, andpreferably, more than 99% by weight, (2) to a degree sufficient toobtain at least 15 residues of N-terminal or internal amino acidsequence by use of a spinning cup sequenator, or (3) to homogeneity bySDS-page under reducing or nonreducing conditions using Coomassie blue,or silver stain. An isolated polypeptide includes the polypeptide insitu within recombinant cells since at least one component of thepolypeptide's natural environment will not be present. Ordinarily,however, an isolated polypeptide will be prepared by a least onepurification step.

As used herein, the term “treatment” refers to both therapeutictreatment and prophylactic or preventative measures. Those in need oftreatment include those already with the disorder as well as those inwhich the disorder is to be prevented.

The phrase “under conditions such that the symptoms are reduced” refersto any degree of qualitative or quantitative reduction in detectablesymptoms of any disease treatable by CD20 binding molecules, includingbut not limited to, a detectable impact on the rate of recovery fromdisease (e.g., rate of weight gain), or the reduction of at least one ofthe symptoms normally associated with the particular disease.

The term “human CD20” (abbreviated herein as hCD20), as used herein, isintended to refer to the human B lymphocyte-restricted differentiationantigen (also known as Bp35). CD20 is expressed during early pre-B celldevelopment and remains until plasma cell differentiation. The CD20molecule may regulate a step in the activation process which is requiredfor cell cycle initiation and differentiation, and is usually expressedat very high levels on neoplastic B cells. CD20 is present on both“normal” B cell as well as “malignant” B cells (i.e. those B cells whoseunabated proliferation can lead to B cell lyphoma).

The terms “affinity”, “binding affinity” and “K_(d)” refer to theequilibrium dissociation constant (expressed in units of concentration)associated with each CD20 binding molecule-CD20 protein complex. Thebinding affinity is directly related to the ratio of the off-rateconstant (generally reported in units of inverse time, e.g., seconds⁻¹)to the on-rate constant (generally reported in units of concentrationper unit time, e.g., molar/second). The binding affinity may bedetermined by, for example, an ELISA assay, kinetic exclusion assay orsurface plasmon resonance. It is noted that certain epitopes can occurrepetitively (multivalent) on a cell surface and that the dissociationconstant (koff) for the binding of an antibody to a repetitive epitopemay be greatly diminished over the dissociation constant for thereaction of the same antibody with the corresponding ligand in univalentform. The diminished dissociation constant arises because when oneantibody-ligand bond dissociates, other bonds hold the bivalent (ormultivalent) antibody to the multivalent ligand, allowing thedissociated bond to form again. The dissociation constant for thereaction between bivalent (or multivalent) Ab and multivalent ligand hasbeen termed the functional affinity to contrast it with intrinsicaffinity, which is the association constant for an antibodiesrepresentative individual site.

The terms “dissociation”, “dissociation rate” and “k_(off)” as usedherein, are intended to refer to the off rate constant for dissociationof a CD20-binding molecule from the antibody/antigen complex.

The terms “association”, “association rate” and “k_(on)” as used herein,are intended to refer to the on rate constant for association of a CD20binding molecule with an antigen to form an antibody/antigen complex.

The terms “effective concentration” and “EC₅₀” as used herein, areintended to refer to the concentration of a CD20 binding moleculecapable of interacting with sufficient quantities of CD20 molecules toproduce an effect on approximately 50% of the treated cells.

DESCRIPTION OF THE INVENTION

The present invention provides CD20 binding molecules and nucleic acidsequences encoding CD20 binding molecules. In particular, the presentprovides CD20 binding molecules with a high binding affinity, and a lowdissociation rate, with regard to human CD20. Preferably, the CD20binding molecules of the present invention comprise light and/or heavychain variable regions with fully human frameworks (e.g. human germlineframeworks). The description of the invention is divided into thefollowing sections below for convenience: I. CD20 Binding Molecules; II.Generating CD20 Binding Molecules; m. Therapeutic Formulations and Uses;and IV. Additional CD20 Binding Molecule Uses.

I. CD20 Binding Molecules

The present invention provides CD20 binding molecules with desirablecharacteristics. In particular, in some embodiments, the CD20 bindingmolecules have a high binding affinity (K_(d)) with regard to humanCD20. In certain embodiments, the CD20 binding molecules have a lowdissociation rate (k_(off)) with regard to human CD20. In preferredembodiments, the CD20α binding molecules of the present invention have ahigh binding affinity, a low dissociation rate and are effective at lowconcentrations. While not necessary to practice or understand theinvention, it is believed that the CD20 binding molecules of the presentinvention, with high binding affinity, and a low dissociation rate, areparticularly well suited for therapeutic use in humans and are lesslikely to elicit a HACA response than other anti-CD20 molecules, such asRITUXAN (C2B8).

In further embodiments, the CD20 binding molecules of the presentinvention bind human CD20. In other embodiments, the CD20 bindingmolecules of the present invention CD20 on the surface of B cells fromCynomolgus macaques.

In preferred embodiments, the CD20 binding molecules of the presentinvention comprise a light and/or heavy chain variable region,preferably having a fully human framework. In particularly preferredembodiments, the CD20 binding molecules of the present inventioncomprise a light and/or heavy chain variable region, preferably having ahuman germline framework. While not necessary to practice or understandthe invention, it is believed that the CD20 binding molecules of thepresent invention (see, e.g. the Examples below) will illicit verylittle or no immunogenic response when administered to a human (e.g. totreat a disease), as the framework regions may be fully human.

As described below in Tables 1 and 2, the present invention providesnumerous CDRs useful for generating CD20 binding molecules. For example,one or more of the CDRs shown can be combined with a frameworksub-region (e.g., a fully human FR1, FR2, FR3, or FR4) in order togenerate a CD20 binding peptide, or a nucleic acid sequence encoding aCD20 binding peptide. Also, the CDRs shown in the Tables below may becombined, for example, such that three CDRs are present in a light chainvariable region, and/or three CDRs are present in a heavy chain variableregion.

The CDRs shown below may be inserted into a human framework (e.g., byrecombinant techniques) into the light and heavy chain frameworks shownin FIGS. 4-5 and 8-9 in order to generate CD20 binding molecules ornucleic acid sequences encoding CD20 binding molecules. For example, theCDRL1 shown in FIG. 4A (or 8A) could be replaced by SEQ ID NO: 1, 3, 5,7, 9, 11, 13, 15, 17, 19, or 21 as shown in Table 1. Likewise, the CDRL1shown in FIG. 4B (or 8B) could be replaced by SEQ ID NO: 2, 4, 6, 8, 10,12, 14, 16, 18, 20, or 22 as shown in Table 1. This same procedure maybe used with all of the CDRs shown in Tables 1-2. Tables 1 and 2 shownimmediately below. TABLE 1 Light Chain CDRs CDR SEQ ID NO Name* SequenceSEQ ID NO:1 CDRL1 RASSSVSYIH SEQ ID NO:2 CDRL1AGGGCCAGCTCAAGTGTAAGTTACATCCAC SEQ ID NO:3 CDRL1 RASSSVHYIH SEQ ID NO:4CDRL1 AGGGCCAGCTCAAGTGTACATTACATCCAC SEQ ID NO:5 CDRL1 RASSSVPYIH SEQ IDNO:6 CDRL1 AGGGCCAGCTCAAGTGTACCGTACATCCAC SEQ ID NO:7 CDRL2 ATSNLAS SEQID NO:8 CDRL2 GCCACATCCAACCTGGCTTCT SEQ ID NO:9 CDRL2 ATTNLAT SEQ IDNO:10 CDRL2 GCCACAACCAACCTGGCTACG SEQ ID NO:11 CDRL2 ATSGLAS SEQ IDNO:12 CDRL2 GCCACATCCGGCCTGGCTTCT SEQ ID NO:13 CDRL2 ATSALAS SEQ IDNO:14 CDRL2 GCCACATCCGCTCTGGCTTCT SEQ ID NO:15 CDRL3 QQWTSNPPT SEQ IDNO:16 CDRL3 CAGCAGTGGACTAGTAACCCACCCACG SEQ ID NO:17 CDRL3 QQWTFNPPT SEQID NO:18 CDRL3 CAGCAGTGGACTTTTAACCCACCCACG SEQ ID NO:19 CDRL3 QQWLSNPPTSEQ ID NO:20 CDRL3 CAGCAGTGGCTGAGTAACCCACCCACT SEQ ID NO:21 CDRL3QTWTFNPPT SEQ ID NO:22 CDRL3 CAGACTTGGACTTTTAACCCTCCCACG*The work of Kabat was used to number residues. CDRs include Kabat andChothia residues.

TABLE 2 Heavy Chain CDRs CDR SEQ ID NO Name* Sequence SEQ ID NO:23 CDRH1GYTFTSYNMH SEQ ID NO:24 CDRH1 GGATACACCTTCACCAGCTACAATATGCAC SEQ IDNO:25 CDRH1 GRTFTSYNMH SEQ ID NO:26 CDRH1 GGCCGTACATTTACCAGTTACAATATGCACSEQ ID NO:27 CDRH2 AIYPGNGDTSYNQKFKG SEQ ID NO:28 CDRH2GCCATCTATCCTGGAAATGGTGATACAAGC TACAATCAGAAGTTCAAAGGC SEQ ID NO:29 CDRH2AIYPGNGDTSYNHKHKG SEQ ID NO:30 CDRH2 GCCATCTATCCTGGAAATGGTGATACAAGCTACAATCATAAGCATAAAGGG SEQ ID NO:31 CDRH2 AIYPGNGDTSYNQKFKW SEQ ID NO:32CDRH2 GCCATCTATCCTGGAAATGGTGATACAAGC TACAATCAGAAGTTTAAATGG SEQ ID NO:33CDRH2 AIYPLNGDTSYNQKFKL SEQ ID NO:34 CDRH2GCTATTTATCCCTTGAATGGTGATACTTCC TACAATCAGAAGTTCAAACTC SEQ ID NO:35 CDRH2AIYPLNGDTSYNRKSKL SEQ ID NO:36 CDRH2 GCTATTTATCCCTTGAATGGTGATACTTCCTACAATCGTAAGTCGAAACTC SEQ ID NO:37 CDRH2 AIYPLTGDTSYNQKFKL SEQ ID NO:38CDRH2 GCTATTTATCCCTTGACGGGTGATACTTCC TACAATCAGAAGTTCAAACTC SEQ ID NO:39CDRH2 AIYPLTGDTSYNQKSKL SEQ ID NO:40 CDRH2GCTATTTATCCCTTGACGGGTGATACTTCC TACAATCAGAAGTCGAAACTC SEQ ID NO:41 CDRH3STYYGGDWYFDV SEQ ID NO:42 CDRH3 TCGACTTACTACGGCGGTGACTGGTACTTC GATGTCSEQ ID NO:43 CDRH3 STYYGGDWQFDV SEQ ID NO:44 CDRH3TCGACTTACTACGGCGGTGACTGGCAGTTC GACGTC SEQ ID NO:45 CDRH3 STYYGGDWQFDESEQ ID NO:46 CDRH3 TCGACTTATTACGGCGGTGACTGGCAGTTC GACGAG SEQ ID NO:47CDRH3 STYYGGDWQFDQ SEQ ID NO:48 CDRH3 TCGACTTATTACGGCGGTGACTGGCAGTTCGACCAG SEQ ID NO:49 CDRH3 STYYGGDWSFDV SEQ ID NO:50 CDRH3TCGACTTACTACGGCGGTGACTGGAGTTTC GATGTC SEQ ID NO:51 CDRH3 STYYGGDWTFDVSEQ ID NO:52 CDRH3 TCGACTTACTACGGCGGTGACTGGACTTTC GATGTC SEQ ID NO:53CDRH3 STYVGGDWTFDV SEQ ID NO:54 CDRH3 TCGACTTACGTGGGCGGTGACTGGACTTTCGATGTC SEQ ID NO:55 CDRH3 SYYVGGDWTFDV SEQ ID NO:56 CDRH3TCGTATTACGTGGGCGGTGACTGGACTTTC GATGTC SEQ ID NO:57 CDRH3 STYVGGDWQFDVSEQ ID NO:58 CDRH3 TCGACTTACGTGGGCGGTGACTGGCAGTTC GATGTC*The work of Kabat was used to number residues. CDRs include both Kabatand Chothia residues.

The present invention also provides sequences that are substantially thesame as the CDR sequences (both amino acid and nucleic acid) shown inthe above Tables. For example, one or two amino acid may be changed inthe sequences shown in the Tables. Also for example, a number ofnucleotide bases may be changed in the sequences shown in the Tables.Changes to the amino acid sequence may be generated by changing thenucleic acid sequence encoding the amino acid sequence. A nucleic acidsequence encoding a variant of a given CDR may be prepared by methodsknown in the art using the guidance of the present specification forparticular sequences. These methods include, but are not limited to,preparation by site-directed (or oligonucleotide-mediated) mutagenesis,PCR mutagenesis, and cassette mutagenesis of an earlier prepared nucleicacid encoding the CDR. Site-directed mutagenesis is a preferred methodfor preparing substitution variants. This technique is well known in theart (see, e.g., Carter et al., (1985) Nucleic Acids Res. 13: 4431-4443and Kunkel et. al., (1987) Proc. Natl. Acad. Sci. USA 82: 488-492, bothof which are hereby incorporated by reference).

Briefly, in carrying out site-directed mutagenesis of DNA, the startingDNA is altered by first hybridizing an oligonucleotide encoding thedesired mutation to a single strand of such starting DNA. Afterhybridization, a DNA polymerase is used to synthesize an entire secondstrand, using the hybridized oligonucleotide as a primer, and using thesingle strand of the starting DNA as a template. Thus, theoligonucleotide encoding the desired mutation is incorporated in theresulting double-stranded DNA.

PCR mutagenesis is also suitable for making amino acid sequence variantsof the starting CDR (see, e.g., Vallette et. al., (1989) Nucleic AcidsRes. 17: 723-733, hereby incorporated by reference). Briefly, when smallamounts of template DNA are used as starting material in a PCR, primersthat differ slightly in sequence from the corresponding region in atemplate DNA can be used to generate relatively large quantities of aspecific DNA fragment that differs from the template sequence only atthe positions where the primers differ from the template.

Another method for preparing variants, cassette mutagenesis, is based onthe technique described by Wells et al., (1985) Gene 34: 315-323, herebyincorporated by reference. The starting material is the plasmid (orother vector) comprising the starting CDR DNA to be mutated. Thecodon(s) in the starting DNA to be mutated are identified. There shouldbe a unique restriction endonuclease site on each side of the identifiedmutation site(s). If no such restriction sites exist, they may begenerated using the above-described oligonucleotide-mediated mutagenesismethod to introduce them at appropriate locations in the startingpolypeptide DNA. The plasmid DNA is cut at these sites to linearize it.A double-stranded oligonucleotide encoding the sequence of the DNAbetween the restriction sites but containing the desired mutation(s) issynthesized using standard procedures, wherein the two strands of theoligonucleotide are synthesized separately and then hybridized togetherusing standard techniques. This double-stranded oligonucleotide isreferred to as the cassette. This cassette is designed to have 5′ and 3′ends that are compatible with the ends of the linearized plasmid, suchthat it can be directly ligated to the plasmid. This plasmid nowcontains the mutated DNA sequence.

Alternatively, or additionally, the desired amino acid sequence encodinga polypeptide variant can be determined, and a nucleic acid sequenceencoding such amino acid sequence variant can be generatedsynthetically. Conservative modifications in the amino acid sequences ofthe CDRs may also be made. Naturally occurring residues are divided intoclasses based on common side-chain properties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilic: cys, ser, thr;

(3) acidic: asp, glu;

(4) basic: asn, gln, his, lys, arg;

(5) residues that influence chain orientation: gly, pro; and

(6) aromatic: trp, tyr, phe.

Conservative substitutions will entail exchanging a member of one ofthese classes for another member of the same class. The presentinvention also provides the complement of the nucleic acid sequencesshown in Tables 1 and 2, as well as nucleic acid sequences that willhybridize to these nucleic acid sequences under low, medium, and highstringency conditions.

The CDRs of the present invention may be employed with any type offramework. Preferably, the CDRs are used with fully human frameworks, orframework sub-regions. In particularly preferred embodiments, theframeworks are human germline sequences. Examples of fully humanframeworks are shown in FIGS. 4-5 and 8-9. Other fully human frameworksor framework sub-regions may also be employed. For example, the NCBI website contains the sequences for the currently known human frameworkregions. Examples of human VH sequences include, but are not limited to,VH1-18, VH1-2, VH1-24, VH1-3, VH1-45, VH1-46, VH1-58, VH1-69, VH1-8,VH2-26, VH2-5, VH2-70, VH3-11, VH3-13, VH3-15, VH3-16, VH3-20, VH3-21,VH3-23, VH3-30, VH3-33, VH3-35, VH3-38, VH3-43, VH3-48, VH3-49, VH3-53,VH3-64, VH3-66, VH3-7, VH3-72, VH3-73, VH3-74, VH3-9, VH4-28, VH4-31,VH4-34, VH4-39, VH4-4, VH4-59, VH4-61, VH5-51, VH6-1, and VH7-81, whichare provided in Matsuda et al., (1998) J. Exp. Med. 188:1973-1975, thatincludes the complete nucleotide sequence of the human immunoglobulinchain variable region locus, herein incorporated by reference. Examplesof human VK sequences include, but are not limited to, A1, A10, A11,A14, A17, A18, A19, A2, A20, A23, A26, A27, A3, A30, A5, A7, B2, B3, L1,L10, L11, L12, L14, L15, L16, L18, L19, L2, L20, L22, L23, L24, L25,L4/18a, L5, L6, L8, L9, O1, O11, O12, O14, O18, O2, O4, and O8, whichare provided in Kawasaki et al., (2001) Eur. J. Immunol. 31:1017-1028;Schable and Zachau, (1993) Biol. Chem. Hoppe Seyler 374:1001-1022; andBrensing-Kuppers et al., (1997) Gene 191:173-181, all of which areherein incorporated by reference. Examples of human VL sequencesinclude, but are not limited to, V1-11, V1-13, V1-16, V1-17, V1-18,V1-19, V1-2, V1-20, V1-22, V1-3, V1-4, V1-5, V1-7, V1-9, V2-1, V2-11,V2-13, V2-14, V2-15, V2-17, V2-19, V2-6, V2-7, V2-8, V3-2, V3-3, V3-4,V4-1, V4-2, V4-3, V4-4, V4-6, V5-1, V5-2, V5-4, and V5-6, which areprovided in Kawasaki et al., (1997) Genome Res. 7:250-261, hereinincorporated by reference. Fully human frameworks can be selected fromany of these functional germline genes. Generally, these frameworksdiffer from each other by a limited number of amino acid changes. Theseframeworks may be used with the CDRs described herein. Additionalexamples of human frameworks which may be used with the CDRs of thepresent invention include, but are not limited to, KOL, NEWM, REI, EU,TUR, TEI, LAY and POM (See, e.g., Kabat et al., (1991) Sequences ofProteins of Immunological Interest, US Department of Health and HumanServices, NIH, USA; and Wu et al., (1970), J. Exp. Med. 132:211-250,both of which are herein incorporated by reference).

Again, while not necessary to practice or understand the invention, itis believed that the reason the use of germline sequences is expected tohelp eliminate adverse immune responses in most individuals is asfollows. Somatic mutations frequently occur in the variable region ofimmunoglobulins as a result of the affinity maturation step that takesplace during a normal immune response. Although these mutations arepredominantly clustered around the hypervariable CDRs, they also impactresidues in the framework regions. These framework mutations are notpresent in the germline genes and are less likely to be immunogenic inpatients. In contrast, the general population has been exposed to thevast majority of framework sequences expressed from germline genes and,as a result of immunologic tolerance, these germline frameworks areexpected to be less, or non-immunogenic in patients. In order tomaximize the likelihood of tolerance, genes encoding the variableregions can be selected from a collection of commonly occurring,functional germline genes, and genes encoding VH and VL regions can befurther selected to match known associations between specific heavy andlight chains of immunoglobulin molecules.

II. Generating CD20 Binding Molecules

In preferred embodiments, the CD20 binding molecules of the presentinvention comprise antibodies or antibody fragments (e.g., comprisingone or more of the CDRs described herein). An antibody, or antibodyfragment, of the present invention can be prepared, for example, byrecombinant expression of immunoglobulin light and heavy chain genes ina host cell. For example, to express an antibody recombinantly, a hostcell may be transfected with one or more recombinant expression vectorscarrying DNA fragments encoding the immunoglobulin light and heavychains of the antibody such that the light and heavy chains areexpressed in the host cell and, preferably, secreted into the medium inwhich the host cell is cultured, from which medium the antibody can berecovered. Standard recombinant DNA methodologies may be used to obtainantibody heavy and light chain genes, incorporate these genes intorecombinant expression vectors and introduce the vectors into hostcells, such as those described in Sambrook, Fritsch and Maniatis (eds),Molecular Cloning; A Laboratory Manual, Second Edition, Cold SpringHarbor, N.Y., (1989), Ausubel, F. M. et al. (eds.) Current Protocols inMolecular Biology, Greene Publishing Associates, (1989) and in U.S. Pat.No. 4,816,397 by Boss et al., all of which are herein incorporated byreference.

To express an antibody with one or more of the CDRs of the presentinvention, DNA fragments encoding the light and heavy chain variableregions are first obtained. These DNAs can be obtained by amplificationand modification of germline light and heavy chain variable sequencesusing the polymerase chain reaction (PCR). Germline DNA sequences forhuman heavy and light chain variable region genes are known in the art(see above).

Once the germline VH and VL fragments are obtained, these sequences canbe mutated to encode one or more of the CDR amino acid sequencesdisclosed herein (see, e.g., Tables 1-2). The amino acid sequencesencoded by the germline VH and VL DNA sequences may be compared to theCDRs sequence(s) desired to identify amino, acid residues that differfrom the germline sequences. Then the appropriate nucleotides of thegermline DNA sequences are mutated such that the mutated germlinesequence encodes the selected CDRs (e.g., the six CDRs that are selectedfrom Tables 1-2), using the genetic code to determine which nucleotidechanges should be made. Mutagenesis of the germline sequences may becarried out by standard methods, such as PCR-mediated mutagenesis (inwhich the mutated nucleotides are incorporated into the PCR primers suchthat the PCR product contains the mutations) or site-directedmutagenesis. In other embodiments, the variable region is synthesized denovo (e.g., using a nucleic acid synthesizer).

Once DNA fragments encoding the desired VH and VL segments are obtained(e.g., by amplification and mutagenesis of germline VH and VL genes, orsynthetic synthesis, as described above), these DNA fragments can befurther manipulated by standard recombinant DNA techniques, for exampleto convert the variable region genes to full-length antibody chaingenes, to Fab fragment genes or to a scFv gene. In these manipulations,a VL- or VH-encoding DNA fragment is operably linked to another DNAfragment encoding another polypeptide, such as an antibody constantregion or a flexible linker. The isolated DNA encoding the VH region canbe converted to a full-length heavy chain gene by operably linking theVH-encoding DNA to another DNA molecule encoding heavy chain constantregions (CH1, CH2 and CH3, see, e.g. FIGS. 10-11). The sequences ofhuman heavy chain constant region genes are known in the art (see e.g.,Kabat, E. A., et al., (1991) Sequences of Proteins of ImmunologicalInterest, Fifth Edition, U.S. Department of Health and Human Services,NIH Publication No. 91-3242) and DNA fragments encompassing theseregions can be obtained by standard PCR amplification. The heavy chainconstant region can be, for example, an IgG1, IgG2, IgG3, IgG4, IgA,IgE, IgM or IgD constant region, but most preferably is an IgG1 or IgG4constant region. For a Fab fragment heavy chain gene, the VH-encodingDNA can be operably linked to another DNA molecule encoding only theheavy chain CH1 constant region. In preferred embodiments, the heavychain constant region is similar to or identical to the constant regionshown in FIG. 11.

The isolated DNA encoding the VL region can be converted to afull-length light chain gene (as well as a Fab light chain gene) byoperably linking the VL-encoding DNA to another DNA molecule encodingthe light chain constant region, CL. The sequences of human light chainconstant region genes are known in the art (see e.g., Kabat, E. A., etal., (1991) Sequences of Proteins of immunological Interest, FifthEdition, U.S. Department of Health and Human Services. NIH PublicationNo. 91-3242) and DNA fragments encompassing these regions can beobtained by standard PCR amplification. The light chain constant regioncan be a kappa or lambda constant region, but most preferably is a kappaconstant region. In preferred embodiments, the light chain constantregion is similar or identical to the constant region shown in FIG. 10.

To create a scFv gene, the VH- and VL-encoding DNA fragments may beoperably linked to another fragment encoding a flexible linker, e.g.,encoding the amino acid sequence (Gly₄-Ser)₃, such that the VH and VLsequences can be expressed as a contiguous single-chain protein, withthe VL and VH regions joined by the flexible linker (see e.g., Huston etal., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; and McCafferty etal., (1990) Nature 348:552-554), all of which are herein incorporated byreference).

To express the antibodies, or antibody fragments of the invention, DNAsencoding partial or full-length light and heavy chains, (e.g. obtainedas described above), may be inserted into expression vectors such thatthe genes are operably linked to transcriptional and translationalcontrol sequences. In this context, the term “operably linked” isintended to mean that an antibody gene is ligated into a vector suchthat transcriptional and translational control sequences within thevector serve their intended function of regulating the transcription andtranslation of the antibody gene. The expression vector and expressioncontrol sequences are generally chosen to be compatible with theexpression host cell used. The antibody light chain gene and theantibody heavy chain gene can be inserted into separate vectors or, moretypically, both genes are inserted into the same expression vector. Theantibody genes may be inserted into the expression vector by standardmethods (e.g., ligation of complementary restriction sites on theantibody gene fragment and vector, or blunt end ligation if norestriction sites are present). Prior to insertion of the light or heavychain sequences, the expression vector may already carry antibodyconstant region sequences. For example, one approach to converting theVH and VL sequences to full-length antibody genes is to insert them intoexpression vectors already encoding heavy chain constant and light chainconstant regions, respectively, such that the VH segment is operablylinked to the CH segment(s) within the vector and the VL segment isoperably linked to the CL segment within the vector. Additionally oralternatively, the recombinant expression vector can encode a signalpeptide that facilitates secretion of the antibody chain from a hostcell. The antibody chain gene can be cloned into the vector such thatthe signal peptide is linked in-frame to the amino terminus of theantibody chain gene. The signal peptide can be an immunoglobulin signalpeptide or a heterologous signal peptide (i.e., a signal peptide from anon-immunoglobulin protein).

In addition to the antibody chain genes, the recombinant expressionvectors of the invention may carry regulatory sequences that control theexpression of the antibody chain genes in a host cell. The term“regulatory sequence” is intended to include promoters, enhancers andother expression control elements (e.g., polyadenylation signals) thatcontrol the transcription or translation of the antibody chain genes.Such regulatory sequences are described, for example, in Goeddel; GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990), herein incorporated by reference. It will beappreciated by those skilled in the art that the design of theexpression vector, including the selection of regulatory sequences maydepend on such factors as the choice of the host cell to be transformed,the level of expression of protein desired, etc. Preferred regulatorysequences for mammalian host cell expression include viral elements thatdirect high levels of protein expression in mammalian cells, such aspromoters and/or enhancers derived from cytomegalovirus (CMV) (such asthe CMV promoter/enhancer), Simian Virus 40 (SV40) (such as the SV40promoter/enhancer), adenovirus, (e.g., the adenovirus major latepromoter (AdMLP)) and polyoma virus. For further description of viralregulatory elements, and sequences thereof, see e.g., U.S. Pat. No.5,168,062 by Stinski, U.S. Pat. No. 4,510,245 by Bell et al. and U.S.Pat. No. 4,968,615 by Schaffner et al., all of which are hereinincorporated by reference.

In addition to the antibody chain genes and regulatory sequences, therecombinant expression vectors of the invention may carry additionalsequences, such as sequences that regulate replication of the vector inhost cells (e.g., origins of replication) and selectable marker genes.The selectable marker gene facilitates selection of host cells intowhich the vector has been introduced (see e.g., U.S. Pat. Nos.4,399,216, 4,634,665 and 5,179,017, all by Axel et al.). For example,typically the selectable marker gene confers resistance to drugs, suchas G418, hygromycin or methotrexate, on a host cell into which thevector has been introduced. Preferred selectable marker genes includethe dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells withmethotrexate selection/amplification) and the neomycin gene (for G418selection).

For expression of the light and heavy chains, the expression vector(s)encoding the heavy and light chains may be transfected into a host cellby standard techniques. The various forms of the term “transfection” areintended to encompass a wide variety of techniques commonly used for theintroduction of exogenous DNA into a prokaryotic or eukaryotic hostcell, e.g., electroporation, calcium-phosphate precipitation,DEAE-dextran transfection and the like.

In certain embodiments, the expression vector used to express the CD20binding molecules of the present invention are viral vectors, such asretro-viral vectors. Such viral vectors may be employed to generatestably transduced cell lines (e.g. for a continues source of the CD20binding molecules). In some embodiments, the GPEX gene productexpression technology (from Gala Design, Inc., Middleton, Wis.) isemployed to generate CD20 binding molecules (and stable cell linesexpressing the CD20 binding molecules). In particular embodiments, theexpression technology described in WO0202783 and WO0202738 to Bleck etal. (both of which are herein incorporated by reference in theirenitirities) is employed.

Preferred mammalian host cells for expressing the recombinant antibodiesof the invention include PER.C6™ cells (Crucell, The Netherlands),Chinese Hamster Ovary (CHO cells) (including dhfr-CHO cells, describedin Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220,used with a DHFR selectable marker, e.g., as described in R. J. Kaufmanand P. A. Sharp (1982) Mol. Biol. 159:601-621), NSO myeloma cells, COScells and SP2 cells. In other preferred embodiments, the host cellsexpress GnT III as described in WO9954342 and U.S. Pat. Pub.20030003097, both herein incorporated by reference, such that expressedCD20 binding molecules have increased ADCC activity. When recombinantexpression vectors encoding antibody genes are introduced into mammalianhost cells, the antibodies are generally produced by culturing the hostcells for a period of time sufficient to allow for expression of theantibody in the host cells or, more preferably, secretion of theantibody into the culture medium in which the host cells are grown.Antibodies can be recovered from the culture medium using standardprotein purification methods.

Host cells can also be used to produce portions of intact antibodies,such as Fab fragments or scFv molecules. It will be understood thatvariations on the above procedure are within the scope of the presentinvention. For example, it may be desirable to transfect a host cellwith DNA encoding either the light chain or the heavy chain of anantibody of this invention. Recombinant DNA technology may also be usedto remove some or all of the DNA encoding either or both of the lightand heavy chains that is not necessary for binding to hCD20. Themolecules expressed from such truncated DNA molecules are alsoencompassed by the antibodies of the invention. In addition,bi-functional antibodies may be produced in which one heavy and onelight chain are an antibody of the invention and the other heavy andlight chain are specific for an antigen other than hCD20 (e.g., bycrosslinking an antibody of the invention to a second antibody bystandard chemical crosslinking methods).

In one preferred system for recombinant expression of an antibody, orfragment thereof, a recombinant expression vector encoding both theantibody heavy chain and the antibody light chain is introduced intodhfr-CHO cells by calcium phosphate-mediated transfection. Within therecombinant expression vector, the antibody heavy and light chain genesare each operably linked to enhancer/promoter regulatory elements (e.g.,derived from SV40, CMV, adenovirus and the like, such as a CMVenhancer/AdMLP promoter regulatory element or an SV40 enhancer/AdMLPpromoter regulatory element) to drive high levels of transcription ofthe genes. The recombinant expression vector may also carry a DHFR gene,which allows for selection of CHO cells that have been transfected withthe vector using methotrexate selection/amplification. The selectedtransformant host cells are cultured to allow for expression of theantibody heavy and light chains and intact antibody is recovered fromthe culture medium. Standard molecular biology techniques are used toprepare the recombinant expression vector, transfect the host cells,select for transformants, culture the host cells and recover theantibody from the culture medium.

In certain embodiments, the antibodies and antibody fragments of thepresent invention are produced in transgenic animals. For example,transgenic sheep and cows may be engineered to produce the antibodies orantibody fragments in their milk (see, e.g., Pollock D P, et al., (1999)Transgenic milk as a method for the production of recombinantantibodies. J. Immunol. Methods 231:147-157, herein incorporated byreference). The antibodies and antibody fragments of the presentinvention may also be produced in plants (see, e.g., Larrick et al.,(2001) Production of secretory IgA antibodies in plants. Biomol. Eng.18:87-94, herein incorporated by reference). Additional methodologiesand purification protocols are provided in Humphreys et al., (2001)Therapeutic antibody production technologies: molecules applications,expression and purification, Curr. Opin. Drug Discov. Devel. 4:172-185,herein incorporated by reference. In certain embodiments, the antibodiesor antibody fragments of the present invention are produced bytransgenic chickens (see, e.g., US Pat. Pub. Nos. 20020108132 and20020028488, both of which are herein incorporated by reference).

III. Therapeutic Formulations and Uses

The CD20 binding molecules of the present invention (e.g. antibodies andantibody fragments) are useful for treating a subject with a disease.The CD20 binding molecules may also be used in diagnostic procedures(e.g. labeled CD20 binding molecules used for tissue imaging). Inpreferred embodiments, the CD20 binding molecules are administered to apatient with B cell lymphoma, which is generally characterized byunabated B cell proliferation.

In some embodiments, the CD20 binding molecules are conjugated tovarious radiolabels for both diagnostic and therapeutic purposes.Radiolabels allow “imaging” of tumors and other tissue, as well helpingto direct radiation treatment to tumors. Exemplary radiolabels include,but are not limited to, ¹³¹I, ¹²⁵I, ¹²³I, ⁹⁹Tc, ⁶⁷Ga, ¹¹¹In, ¹⁸⁸Re,¹⁸⁶Re, and preferably, ⁹⁰Y.

In certain embodiments, the disease treated is Non-Hodgkin's lymphoma(NHL). In some embodiments, the disease is selected from relapsedHodgkin's disease, resistant Hodgkin's disease high grade, low grade andintermediate grade non-Hodgkin's lymphomas (NHLs), B cell chroniclymphocytic leukemia (B-CLL), lymphoplasmacytoid lymphoma (LPL), mantlecell lymphoma (MCL), follicular lymphoma (FL), diffuse large celllymphoma (DLCL), Burkitt's lymphoma (BL), AIDS-related lymphomas,monocytic B cell lymphoma, angioimmunoblastic lymphoadenopathy, smalllymphocytic; follicular, diffuse large cell; diffuse small cleaved cell;large cell immunoblastic lymphoblastoma; small, non-cleaved; Burkift'sand non-Burkitt's; follicular, predominantly large cell; follicular,predominantly small cleaved cell; follicular, mixed small cleaved andlarge cell lymphomas, and systemic lupus erythematosus (SLE). Inparticular embodiments, the disease treated is Waldenstrom'sMacroglobulinemia (WM) or Chronic Lymphocytic Leukemia (CLL).

In some embodiments, the CD20 binding molecules of the present inventionare used for treatment of diseases wherein depletion of CD20+ cells istherapeutically beneficial, such as Waldenstrom's macroglobulianemia,multiple myeloma, plasma cell dyscrasias, chronic lymphocytic leukemia,treatment of transplant, hairy cell leukemia, ITP, Epstein Barr viruslymphomas after stem cell transplant, and Kidney transplant, see U.S.Pat. Pub. 20020128448, herein incorporated by reference. In otherembodiments, the CD20 binding molecules of the present invention areused for the treatment of a disease selected from the group consistingof B cell lymphomas, leukemias, myelemas, autoimmune disease,transplant, graft-vs-host disease, infectious diseases involving Bcells, lymphoproliferation diseases, and treatment of any disease orcondition wherein suppression of B cell activity and/or humoral immunityis desirably suppressed. In certain embodiments, the CD20 bindingmolecules of the present invention are used for the treatment of adisease selected from the group consisting of B cell lymphomas,leukemia, myelema, transplant, graft-vs-host disease, autoimmunedisease, lymphoproliferation conditions, and other treatment diseasesand conditions wherein the inhibition of humoral immunity, B cellfunction, and/or proliferation, is therapeutically beneficial. Infurther embodiments, the CD20 binding molecules of the present inventionare used for the treatment of B-ALL, Hairy cell leukemia, Multiplemyeloma, Richter Syndrome, Acquired Factor VIII inhibitors,Antiphospholipid syndrome, Autoimmune hemolytic anemia, Autoimmunethrombocytopenia, Bullous pemphigoid, Cold hemagglutinin disease, Evan'sSyndrome, Goodpasture's syndrome, Idiopathic membranous nephropathy,Idiopathic thrombocytopenic purpura, IgM associated polyneuropathy,Kaposi sarcoma-associated herpesvirus (KSHV)-related multicentricCastleman disease (MCD), Myasthenia gravis, Pemphigus vulgaris, Primarybiliary cirrhosis, Pure red cell aplasia, Rheumatoid arthritis,Sjogren's Syndrome, Systemic immune complex vasculitis, Systemic lupuserythematosus, Type II mixed cryoglobulinemia, Wegener's granulomatosis,Allograft rejection, Post-transplant lymphoproliferative disease, orPurging of stem cells for bone marrow transplantation.

In preferred embodiments, the subject treat is not immunosuppressed(e.g. an SLE patient). While not limited to any mechanism, it is believethat the CD20 binding molecules of the present invention are less likelyto illicit a HACA response than previously known anti-CD20 antibodies,especially in non-immunosuppressed patients. As such,non-immunosuppressed patients can be treated without the overridingconcern for an adverse HACA reaction. Also, with the improved bindingability of the CD20 binding molecules of the present invention, lowerdoses may be administered to patients (further avoiding the risk of aHACA response), or higher doses may be administered without risking alife threatening HACA response. In other preferred embodiments, thesubject has rheumatoid arthritis or other autoimmune disease (See, e.g.,Edwards et al., Rheumatology (Oxford) 2001 February; 40(2):205-11,herein incorporated by reference).

The CD20 binding molecules of the present invention may also beadministered in combination with other therapeutic moieties. Forexample, the CD20 binding molecules may be administered a part of achemotherapeutic program (e.g. CHOP). The CD20 binding molecules mayalso be administered with cytokines, G-CSF, or IL-2 (See, U.S. Pat. No.6,455,043, herein incorporated by reference).

The CD20 binding molecules of the present invention may be administeredby any suitable means, including parenteral, non-parenteral,subcutaneous, topical, intraperitoneal, intrapulmonary, intranasal, andintralesional administration (e.g., for local immunosuppressivetreatment). Parenteral infusions include, but are not limited to,intramuscular, intravenous, intra-arterial, intraperitoneal, orsubcutaneous administration. In addition, CD20 binding molecules aresuitably administered by pulse infusion, particularly with decliningdoses. Preferably, the dosing is given by injections, most preferablyintravenous or subcutaneous injections, depending in part on whether theadministration is brief or chronic.

Dosage regimens may be adjusted to provide the optimum desired response(e.g., a therapeutic or prophylactic response). For example, a singlebolus may be administered, several divided doses may be administeredover time or the dose may be proportionally reduced or increased asindicated by the exigencies of the therapeutic situation. It isadvantageous to formulate parenteral compositions in dosage unit formfor ease of administration and uniformity of dosage. The dosages of theCD20 binding molecules of the present invention are generally dependenton (a) the unique characteristics of the active compound and theparticular therapeutic or prophylactic effect to be achieved, and (b)the limitations inherent in the art of compounding such an activecompound for the treatment of sensitivity in individuals.

An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of an antibody or antibody fragment(or other CD20 binding molecule of the invention) is 0.1-20 mg/kg, morepreferably 1-10 mg/kg. In some embodiments, the dosage is from 50-600mg/m² (e.g. 375 mg/m²). It is to be noted that dosage values may varywith the type and severity of the condition to be alleviated. It is tobe further understood that for any particular subject, specific dosageregimens should be adjusted over time according to the individual needand the professional judgment of the person administering or supervisingthe administration of the compositions, and that dosage ranges set forthherein are exemplary only and are not intended to limit the presentinvention.

The dosage administered will, of course, vary depending upon knownfactors such as the pharmacodynamic characteristics of the particularagent, its mode and route of administration, the age, health, and weightof the recipient, the nature and extent of symptoms, the kind ofconcurrent treatment, the frequency of treatment, and the effectdesired. For example, a daily dosage of active ingredient can be about0.01 to 100 milligrams per kilogram of body weight. Ordinarily 1.0 to 5,and preferably 1 to 10 milligrams per kilogram per day given in divideddoses 1 to 6 times a day or in sustained release form, may be effectiveto obtain desired results.

The CD20 binding molecules of the invention can be incorporated intopharmaceutical compositions suitable for administration to a subject.For example, the pharmaceutical composition may comprise a CD20 bindingmolecule (e.g. an antibody or antibody fragment) and a pharmaceuticallyacceptable carrier. As used herein, “pharmaceutically acceptablecarrier” includes solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike that are physiologically compatible. Examples of pharmaceuticallyacceptable carriers include one or more of the following: water, saline,phosphate buffered saline, dextrose, glycerol, ethanol and the like, aswell as combinations thereof. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition.Pharmaceutically acceptable carriers may further comprise minor amountsof auxiliary substances such as wetting or emulsifying agents,preservatives or buffers, which enhance the shelf life or effectivenessof the CD20 binding molecules.

The compositions of this invention may be in a variety of forms. Theseinclude, for example, liquid, semi-solid and solid dosage forms, such asliquid solutions (e.g., injectable and infusible solutions), dispersionsor suspensions, tablets, pills, powders, liposomes and suppositories.The preferred form depends on the intended mode of administration andtherapeutic application. Typical preferred compositions are in the formof injectable or infusible solutions, such as compositions similar tothose used for passive immunization of humans with other antibodies.

Therapeutic compositions typically are sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, dispersion, liposome, or other orderedstructure suitable to high drug concentration. Sterile injectablesolutions can be prepared by incorporating the active compound (i.e.,antibody or antibody fragment) in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asrequired, followed by sterile filtration. Generally, dispersions areprepared by incorporating the active compound into a sterile vehiclethat contains a basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, the preferredmethods of preparation are vacuum drying and freeze-drying that yield apowder of the active ingredient plus any additional desired ingredientfrom a previously sterile-filtered solution thereof. The proper fluidityof a solution can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. Prolongedabsorption of injectable compositions can be brought about by includingin the composition an agent that delays absorption, for example,monostearate salts and gelatin.

In certain embodiments, the active compound may be prepared with acarrier that will protect the compound against rapid release, such as acontrolled release formulation, including implants, transdermal patches,and microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art (see, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson. ed., MarcelDekker, Inc., New York, 1978).

In certain embodiments, the CD20 binding molecules of the invention maybe orally administered, for example, with an inert diluent or anassimilable edible carrier. The compound (and other ingredients, ifdesired) may also be enclosed in a hard or soft shell gelatin capsule,compressed into tablets, or incorporated directly into the subject'sdiet. For oral therapeutic administration, the compounds may beincorporated with excipients and used in the form of ingestible tablets,buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers,and the like. To administer a compound of the invention by other thanparenteral administration, it may be necessary to coat the compoundwith, or co-administer the compound with, a material to prevent itsinactivation.

The pharmaceutical compositions of the invention may include a“therapeutically effective amount” or a “prophylactically effectiveamount” of an antibody or antibody fragment of the invention. A“therapeutically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredtherapeutic result. A therapeutically effective amount of the antibodyor antibody fragment may vary according to factors such as the diseasestate, age, sex, and weight of the individual, and the ability of theantibody or antibody fragment to elicit a desired response in theindividual. A therapeutically effective amount is also one in which anytoxic or detrimental effects of the antibody or antibody fragment areoutweighed by the therapeutically beneficial effects. A“prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically, since a prophylactic dose is used insubjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

IV. Additional CD20 Binding Molecule Uses

CD20 binding molecules of the present invention, such as anti-CD20peptides and/or antibodies are useful for immunoassays which detect orquantify CD20 in a sample or B cells bearing CD20. An immunoassay forCD20 typically comprises incubating a biological sample in the presenceof a detectably labeled high affinity anti-CD20 peptide and/or antibodyof the present invention capable of selectively binding to CD20, anddetecting the labeled peptide or antibody which is bound in a sample.Various clinical assay procedures are well known in the art.

Thus, an anti-CD20 peptide or antibody, can be captured onnitrocellulose, or on any other solid support which is capable ofimmobilizing soluble proteins. A CD20-containing sample is then added tothe support which is subsequently washed with suitable buffers to removeunbound proteins. A second, detectably labeled, CD20 specific peptide orantibody is added to the solid phase support that can then be washedwith the buffer a second time to remove unbound detectably labeledpeptide or antibody. The amount of bound label on the solid support canthen be detected by known methods.

By “solid phase support” or “carrier” is intended any support capable ofbinding peptide, antigen or antibody. Well-known supports or carriers,include glass, polystyrene, polypropylene, polyethylene, dextran, nylon,amylases, natural and modified celluloses, polyacrylamides, agaroses,and magnetite. The nature of the carrier can be either soluble to someextent or insoluble for the purposes of the present invention. Thesupport material can have virtually any possible structuralconfiguration so long as the coupled molecule retains its ability tobind to CD20. Thus, the support configuration can be spherical, as in abead, or cylindrical, as in the inside surface of a test tube, or theexternal surface of a rod. Alternatively, the surface can be flat suchas a sheet, culture dish, test strip, microtiter plates, etc. Preferredsupports include polystyrene beads. Those skilled in the art will knowmany other suitable carriers for binding antibody, peptide or antigen,or can ascertain the same by routine experimentation. Well known methodscan be used to determine the binding activity of a given lot ofanti-CD20 peptide and/or antibody. Those skilled in the art candetermine operative and optimal assay conditions by routineexperimentation.

Detectably labeling a CD20 binding molecule, such as a CD20-specificpeptide and/or antibody, can be accomplished by coupling to an enzymefor use in an enzyme immunoassay (EIA), or enzyme-linked immunosorbentassay (ELISA). The linked enzyme reacts with the exposed substrate togenerate a chemical moiety which can be detected, for example, byspectrophotometric, fluorometric or by visual means. Enzymes which canbe used to detectably label the CD20 binding molecules of the presentinvention include, but are not limited to, malate dehydrogenase,staphylococcal nuclease, delta-5-steroid isomerase, yeast alcoholdehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphateisomerase, horseradish peroxidase, alkaline phosphatase, asparaginase,glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase.

By radioactively labeling the CD20 binding molecules, it is possible todetect CD20 through the use of a radioimmunoassay (RIA) (see, forexample, Work, et al., (1978) Laboratory Techniques and Biochemistry inMolecular Biology, North Holland Publishing Company, N.Y.). Theradioactive isotope can be detected by such means as the use of a gammacounter or a scintillation counter or by autoradiography.

It is also possible to label the CD20 binding molecules with afluorescent compound. When the fluorescently labeled molecule is exposedto light of the proper wave length, its presence can then be detecteddue to fluorescence. Among the most commonly used fluorescent labelingcompounds are fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

The CD20 binding molecules can also be detectably labeled usingfluorescence-emitting metals such as ¹²⁵Eu, or others of the lanthanideseries. These metals can be attached to the CD20 binding molecule usingsuch metal chelating groups as diethylenetriaminepentaacetic acid (DTPA)or ethylenediamine-tetraacetic acid (EDTA).

The CD20 binding molecules also can be detectably labeled by coupling toa chemiluminescent compound. The presence of the chemiluminescentlylabeled molecule is then determined by detecting the presence ofluminescence that arises during the course of a chemical reaction.Examples of particularly useful chemiluminescent labeling compounds areluminol, isoluminol, theromatic acridinium ester, imidazole, acridiniumsalt and oxalate ester.

Likewise, a bioluminescent compound can be used to label the CD20binding molecules of the present invention. Bioluminescence is a type ofchemiluminescence found in biological systems in which a catalyticprotein increases the efficiency of the chemiluminescent reaction. Thepresence of a bioluminescent protein is determined by detecting thepresence of luminescence. Important bioluminescent compounds forpurposes of labeling are luciferin, luciferase and aequorin.

Detection of the CD20 binding molecules can be accomplished by ascintillation counter, for example, if the detectable label is aradioactive gamma emitter, or by a fluorometer, for example, if thelabel is a fluorescent material. In the case of an enzyme label, thedetection can be accomplished by colorometric methods which employ asubstrate for the enzyme. Detection can also be accomplished by visualcomparison of the extent of enzymatic reaction of a substrate tosimilarly prepared standards.

In some embodiments of the present invention, the CD20 which is detectedby the above assays can be present in a biological sample. Any samplecontaining CD20 can be used. Preferably, the sample is a biologicalfluid such as, for example, blood, serum, lymph, urine, cerebrospinalfluid, amniotic fluid, synovial fluid, a tissue extract or homogenate,and the like. However, the invention is not limited to assays using onlythese samples, as it is possible for one of ordinary skill in the art todetermine suitable conditions which allow the use of other samples.

In situ detection can be accomplished by removing a histologicalspecimen from a patient, and providing the combination of labeled CD20binding molecules of the present invention to such a specimen. The CD20binding molecule is preferably provided by applying or by overlaying thelabeled CD20 binding molecule to a biological sample. Through the use ofsuch a procedure, it is possible to determine not only the presence ofCD20 but also the distribution of CD20 in the examined tissue. Using thepresent invention, those of ordinary skill will readily perceive thatany of a wide variety of histological methods (such as stainingprocedures) can be modified in order to achieve such in situ detection.

The CD20 binding molecules of the present invention can be adapted forutilization in an immunometric assay, also known as a “two-site” or“sandwich” assay. In a typical immunometric assay, a quantity ofunlabeled CD20 binding molecule (such as an anti-CD20 antibody) is boundto a solid support that is insoluble in the fluid being tested and aquantity of detectably labeled soluble antibody is added to permitdetection and/or quantitation of the ternary complex formed betweensolid-phase antibody, antigen, and labeled antibody.

Typical, and preferred, immunometric assays include “forward” assays inwhich the CD20 binding molecule (e.g. antibody) bound to the solid phaseis first contacted with the sample being tested to extract the CD20 fromthe sample by formation of a binary solid phase antibody-CD20 complex.After a suitable incubation period, the solid support is washed toremove the residue of the fluid sample, including unreacted CD20, ifany, and then contacted with the solution containing a known quantity oflabeled antibody (which functions as a “reporter molecule”). After asecond incubation period to permit the labeled antibody to complex withthe CD20 bound to the solid support through the unlabeled antibody, thesolid support is washed a second time to remove the unreacted labeledantibody. This type of forward sandwich assay can be a simple “yes/no”assay to determine whether CD20 is present or can be made quantitativeby comparing the measure of labeled antibody with that obtained for astandard sample containing known quantities of CD20.

Other types of “sandwich” assays, which can also be useful with CD20,are the so-called “simultaneous” and “reverse” assays. A simultaneousassay involves a single incubation step wherein the antibody bound tothe solid support and labeled antibody are both added to the samplebeing tested at the same time. After the incubation is completed, thesolid support is washed to remove the residue of fluid sample anduncomplexed labeled antibody. The presence of labeled antibodyassociated with the solid support is then determined as it would be in aconventional “forward” sandwich assay. In the “reverse” assay, stepwiseaddition first of a solution of labeled antibody to the fluid samplefollowed by the addition of unlabeled antibody bound to a solid supportafter a suitable incubation period, is utilized. After a secondincubation, the solid phase is washed in conventional fashion to free itof the residue of the sample being tested and the solution of unreactedlabeled antibody. The determination of labeled antibody associated witha solid support is then determined as in the “simultaneous” and“forward” assays.

In some embodiments, the CD20 binding molecules of this invention,attached to a solid support, can be used to remove CD20 (or B cellsbearing CD20) from fluids or tissue or cell extracts. In a preferredembodiment, they are used to remove CD20 from blood or blood plasmaproducts. In another preferred embodiment, the CD20 binding moleculesare advantageously used in extracorporeal immunoadsorbent devices, whichare known in the art (see, for example, Seminars in Hematology, 26 (2Suppl. 1)(1989)). Patient blood or other body fluid is exposed to theattached CD20 binding molecule, resulting in partial or complete removalof circulating CD20 (free or in immune complexes), following which thefluid is returned to the body. This immunoadsorption can be implementedin a continuous flow arrangement, with or without interposing a cellcentrifugation step. See, for example, Terman, et al., (1976) J.Immunol. 117:1971-1975.

Experimental

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: M (molar); mM (millimolar); nM (nanomolar); pM(picomolar); mg (milligrams); μg (micrograms); pg (picograms); ml(milliliters); μl (microliters); ° C. (degrees Celsius); OD (opticaldensity); nm (nanometer); BSA (bovine serum albumin); and PBS(phosphate-buffered saline solution).

EXAMPLE 1 CD20 Binding Molecules

This example describes how certain, exemplary, CD20 binding moleculesmay be constructed. In particular, this example describes how elevendifferent CD20 binding molecules (AME 21E1 Hum, AME 6F1, AME 2C2, AME1D10, AME 15, AME 18, AME 33, AME 5-3, AME 1C2, AME 4H5, and AME 5) maybe constructed, and expressed, for example, as Fabs or full IgGs.

The light and heavy chain variable regions of the eleven different CD20binding molecules may be constructed as follows. The collection of 6CDRs for each of the CD20 binding molecules is shown in Table 3, withthe sequence ID number for the amino acid sequence listed first andsequence ID number for the nucleic acid sequence listed second. TABLE 3Light and Heavy Chain CDRs in Exemplary CD20 Binding Molecules Anti-CD20CDRL1 CDRL2 CDRL3 CDRH1 CDRH2 CDRH3 Molecule SEQ ID NO: SEQ ID NO: SEQID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: AME 21E1 Hum 1 and 2 7 and 8 19and 20 23 and 24 27 and 28 49 and 50 AME 6F1 1 and 2 7 and 8 19 and 2023 and 24 27 and 28 51 and 52 AME 2C2 1 and 2 7 and 8 19 and 20 23 and24 33 and 34 53 and 54 AME 1D10 5 and 6 7 and 8 19 and 20 23 and 24 33and 34 53 and 54 AME 15 5 and 6 7 and 8 19 and 20 23 and 24 35 and 36 53and 54 AME 18 5 and 6 13 and 14 19 and 20 25 and 26 37 and 38 55 and 56AME 33 5 and 6 13 and 14 19 and 20 25 and 26 39 and 40 57 and 58 AME 5-31 and 2 7 and 8 17 and 18 23 and 24 27 and 28 43 and 44 AME 1C2 3 and 4 9 and 10 17 and 18 23 and 24 29 and 30 45 and 46 AME 4H5 3 and 4 7 and8 17 and 18 23 and 24 31 and 32 47 and 48 AME 5 3 and 4 11 and 12 21 and22 23 and 24 31 and 32 45 and 46

The CDRs in Table 3 may be combined with any framework, such as a humangermline framework, in order to generate variable regions (which may,for example, be expressed as Fvs). For example, in order to generate thevariable regions for the eleven anti-CD20 molecules named in thisexample, the CDRs in table 3 are combined with human germlineframeworks, as shown in Table 4. TABLE 4 Human Germline Frameworks Usedto Generate Named CD20 Binding Molecules Anti-CD20 Molecule VL FrameworkVH Framework AME 5-3 VkI (DPK4) (A20) VHI DP7/21-2 AME 1C2 VkI (DPK4)(A20) VHI DP7/21-2 AME 4H5 VkI (DPK4) (A20) VHI DP7/21-2 AME 5 VkI(DPK4) (A20) VHI DP7/21-2 AME 21E1 Hum VkIII (A27) (DPK22) VH5-51(DP-73) AME 6F1 VkIII (A27) (DPK22) VH5-51 (DP-73) AME 2C2 VkIII (A27)(DPK22) VH5-51 (DP-73) AME 1D10 VkIII (A27) (DPK22) VH5-51 (DP-73) AME15 VkIII (A27) (DPK22) VH5-51 (DP-73) AME 18 VkIII (A27) (DPK22) VH5-51(DP-73) AME 33 VkIII (A27) (DPK22) VH5-51 (DP-73)

The sequences for the full light and heavy chain variable regions of twoof the eleven CD20 binding molecules is provided in FIGS. 2-3 and 6-7.In particular, FIGS. 2 and 3 show the amino acid and nucleic acidsequences for the light and heavy chain variable regions of AME 33(which, together, provides the full Fv sequence for AME 33). FIGS. 6 and7 show the amino acid and nucleic acid sequences for the light and heavychain variable regions of AME 5.

The light and heavy chain variable regions for the eleven CD20 bindingmolecules discussed in this example may be combined with light and heavychain constant regions and expressed as Fabs or full antibodies (e.g.IgG). These sequences are preferably linked to a leader sequence (e.g. aleader sequence at the beginning of the light chain sequence). Oneexample of leader sequence that may be employed is METPAQLLFLLLLWLPDTTG(SEQ ID NO:105). Other leader sequences may be employed. Also, any humanconstant region allotype chain may be employed. For example, FIGS. 10and 11 show the complete light and heavy chains for AME 33, whichinclude the light and heavy chain constant regions. These figures mayalso be the source of the constant regions used to make the Fab and IgGsof the other CD20 binding molecules named in this example. It is notedthat the constant regions are underlined in FIGS. 10A and 11A. Also, theanti-CD20 molecules in this example may alternatively employ the heavychain constant regions shown in FIGS. 10 and 11, except with an aminoacid substitution in the Fc region. In particular, the heavy chainconstant region shown in FIG. 11 may contain a D280H mutation or a K290Smutation (FIG. 11A shows positions 280 and 290 in bold, without themutations). FIG. 11B shows a bold and underlined “GAC.” This “GAC” maybe changed to “CAT” in order to encode the D280H mutation.

In order to express the anti-CD20 binding molecules in this example (asFabs or IgGs), procedures known in the art may be used. For example, theeleven CD20 binding molecules in this example can be expressed, as Fabsor IgG's, in mammalian expression systems (or bacterial, fungal andplant expression systems) using either a single vector or double vectorsystem. In a single vector system both heavy and light chains aremanufactured or cloned within an expression cassette, which contains allrequired regulatory elements for expression. A double vector systemsimply has these two expression cassettes in separate plasmids. Eitherthe single, or combined plasmids in the double vector system, may betransfected into a host cell line such as Chinese Hamster Ovary (CHO)cells or the retinal cell line PerC6, selected for, expanded andcultured to express the Fab or IgG proteins as is known in the art (seeAntibody Expression and Engineering: Developed from a SymposiumSponsored by the Division of Biochemical Technology at the 207thNational Meeting of the American Chemical Society, San die [AcsSymposium Series, 604]).

Fabs may also be expressed in a bacterial expression system, as this isless time consuming and less expensive than mammalian systems. Here Fabscan be inserted and expressed within a M13 viral expression system.Bacterial expressed Fabs are secreted and also accumulate within theperiplamic space between the bacterial cell wall and its cell membrane.The Fab can be released from this periplasmic space by a number oftechniques including hypotonic shock and freeze thaw procedures commonin the art. Fab's can also be generated from intact IgG by proteolyticcleavage using a protease such as papain. The Fab portion of thecleavage product can then be purified away from the Fc portion of thecleavage product. Fabs and IgG's can be purified with any variety ofchromatographic and specific adsorption techniques that are also knownin the art (see Antibodies: A Laboratory Manual, by Ed Harlow (Editor),David Lane (Editor), Cold Spring Harbor Press). For example IgG's can beeasily purified from cellular supernatants by specific binding usingrProtein A affinity chromatography followed by Mono S cation exchangechromatography.

EXAMPLE 2 Fixed Ramos Cell ELISA

This example describes a fixed Ramos cell ELISA binding assay with CD20binding molecules. In particular, this example assayed AME 4H5, AME 15,AME 18, AME 33 and AME 1D10 expressed as Fab and IgG to determineoverall binding, off-rate, and on-rate. This example also tested inhouse C2B8 fabs and full antibody, as well as commercial RITUXAN(Oncology Supply Co.) in the same assay for comparison purposes. TheC2B8 antibody is deposited with the ATCC as deposit number 69119.

Ramos cells (ATCC) were grown in RPMI 1640 containing 10% heatinactivated Fetal Bovine Serum. Fifty microliters of Ramos cells(2-4×10⁶/ml), were pipetted into each well of a 96 well Poly-D-Lysinecoated plate (BIOCAT Becton Dickinson Labware) and incubated at 37° C.,5% CO₂ for 18 hours. Media was gently aspirated and 100 ml of aqueousbuffered zinc formalin (Anatech, Ltd.) was added to each well for 15minutes at room temperature. Z-fix was removed and the plate was washedwith phosphate-buffered saline (PBS) containing 0.05% Tween 20. Theplate was blocked for 1 hour with 1% bovine serum albumin (BSA) in PBS.Dilutions of Fab or IgG were incubated with the fixed and blocked Ramoscells for 1 hour at room temperature. The plate was washed 3× with PBS0.05% Tween 20, and 50 ml of anti-His6 Peroxidase Mouse MonoclonalAntibody (Roche Diagnostics Corporation) at a 1:500 dilution were addedto the wells and incubated 1 hour at room temperature. The plate waswashed as above and developed with tetramethyl benzidine, and thereaction was stopped with 5N H₂SO₄. The plate absorbance was read at OD450 nm. Dilutions of Fab and conjugate were in 1% BSA/PBS.

IgG assays were performed similarly except IgG bound was detected withgoat anti-human IgG-HRP. To compare antibody off-rate, after incubationwith Fab or IgG the plates were incubated over night in PBS/BSA, beforeproceeding with the second antibody step. To compare antibody on-rate,incubation steps for Fab/IgG binding and with the second antibody wereboth of 5 minutes duration.

The results from this example are presented in FIGS. 12 and 13. As shownin these figures, overall binding of Fabs AME 4H5, 15, 18, 33 and 1D10are more effective than C2B8 Fab. The dose-response curve for the AMEantibodies is shifted to the left relative to the C2B8 antibody andcommercial RITUXAN, and the maximum OD attained is approximately 1.5times that seen with the C2B8 antibody. In regard to off-rate, Fabs ofAME 4H5, 15, 18, 33 and 1D10 show a dramatically decreased off-raterelative to C2B8 Fab (FIG. 12B). The off-rate for AME antibodiesexpressed as whole IgGs is similar to that of the C2B8 antibody andcommercial RITUXAN (FIG. 13A). In regard to on-rate, Fabs of AME 4H5,15, 18, 33 and 1D10 show an on-rate similar to that of the C2B8 Fab,with increased overall binding (maximum OD attained) (FIG. 12C). Thedose-response for AME antibodies expressed as whole IgGs is shifted tothe left of that of the full C2B8 antibody (FIG. 13B).

EXAMPLE 3 Immunofluorescent Live B Lymphoma Binding Assays

This example describes immunofluorescent live B lymphoma binding assays.In particular, Fabs of AME 33, AME 5 and C2B8 were assayed as describedbelow.

Fab Staining of PBMC's for CD20 FACS Analysis

Peripheral blood mononuclear cells (PBMC) were isolated from normalhuman blood by flotation on Ficol-Hypaque (Sigma-1077). Cells werecounted and resuspended in PBS+1% BSA to give 2-5×10⁶ cells/ml. Onehundred microliters of diluted cells were dispensed into polystyrenetubes (Falcon #2058) and anti-CD20 Fab antibodies diluted in PBS+1% BSAwere added. Tubes were incubated 1 hour at room temperature. Fourmilliliters of PBS+1% BSA were added to each tube and the tubes werecentrifuged at 300 xG for 10 minutes. The supernatant was removed andthe cells resuspended in 100 μl PBS+1% BSA. Anti-Penta-His AlexaFluor488 conjugate (Qiagen #35310), 2 μl per tube, was added, the tubes weremixed and incubated for 1 hour in the dark at room temperature. Sampleswere washed as previously described. The supernatant was removed and thecells resuspended in PBS+1% BSA+2 μg/ml Propidium iodide. Fluoresencewas analyzed on a Becton Dickinson FACScan or FACSort flow cytometer anddata analyzed using Cell Quest (Becton Dickinson) or WinMDI software.

The results are presented in FIG. 14. Results for Daudi cells are shownin FIG. 14A, results for Wil2-S cells are shown in FIG. 14B, and resultsfor Ramos cells are shown in FIG. 14C. As shown in this figure, Fabs ofAME 5 and 33 are more effective in binding to live B lymphoma cell linesthan C2B8. It is noted that the dose response curve for AME 5 and AME 33is shifted to the left relative to C2B8.

EXAMPLE 4 IgG Binding Measured by KinExA

This example describes assays for measuring Kd, Kon, and Koff of variousCD20 IgGs. In particular, this assay tested AME 33, AME 5, AME 6F1, andthe C2B8 antibody for their binding to SKW 6.4 B lymphoma cells anddetermined K_(d), Kon, and Koff for each of these molecules with the aidof KinExa equilibrium software. In addition, AME 33 and C2B8 were testedfor binding to primary human peripheral blood B cells.

Kd Measurement:

SKW 6.4 cells were grown in DMEM media supplemented with 10% FCS andharvested at 5-10×10⁵ cells per ml. The cells were washed with 5 volumesof PBS and resuspended in PBS with 1% BSA at approx. 1×10⁸ cells per ml.For human peripheral blood B cells, fresh CD19 positively sorted B cellswere obtained from Allcells. These cells were washed two times in PBSwith 1% BSA and resuspended at a concentration of 8×10⁷/ml.

Twelve, 3 fold serial dilutions were then made and 100 ul of eachdilution placed in a 96 well plate. To each dilution 100 ml of antibodyat 100 ng/ml was added and the plate incubated at 37° C., 5% CO₂ for 4hours. Each sample was then filtered with a 96 well 1 micron filter(Millipore) to remove the cells and bound antibody. Free antibody insolution was then quantified using an ELISA. Briefly, free IgG wascaptured in a 96 well plate coated with 1 mg/ml of anti-human kappaantibody (Southern Biotechnology) and detected using an anti-human Fcantibody coupled with HRP (Southern Biotechnology). The plate wasdeveloped using Fast TMB substrate (Pierce) and read at 450 nm.

The Kd of the antibody was then determined using the KinExA equilibriumsoftware for an unknown antigen concentration. The OD450 value for eachserial dilution was fitted with an estimated antigen concentration andthe actual antibody concentration. The Kd and actual antigenconcentration were then calculated. The results for the SKW 6.4 cellassays are shown in Table 5. These results show that AME 33 and AME 5have a Kd that is 10-fold enhanced compared to the C2B8 antibody. Theresults for the primary human peripheral blood B cells show a Kd for AME33 of 113 pM and a Kd for C2B8 of 1500 pM. Again, AME 33 is shown tohave a Kd greater than 10-fold (almost fifteen fold) that of the C2B8antibody. TABLE 5 AME 33 AME 5 AME 6F1 C2B8 Kd; pM 97 145 2500 2097 Kon;×10⁵ M⁻¹, s⁻¹ 7.8 5.8 1.0 4.7 Koff; ×10⁻⁵ s⁻¹ 7.7 7.9 25.0 104.5Kinetic Measurements:

SKW 6.4 cells were grown in DMEM media supplemented with 10% FCS andharvested at 5-10×10⁵ cells per ml. The cells were washed with 5 volumesof PBS and resuspended in PBS with 1% BSA at approx. 1×10⁷ cells per mland kept in a water bath at 37° C. An equal volume of antibody at 100ng/ml in PBS with 1% BSA at 37° C. was then added to the cells and thetiming for the experiment started. At time intervals from 60 seconds to10800 seconds a sample of the antibody cell solution was sampled andfiltered to remove cells and bound antibody. Free antibody remaining ateach time point was then quantified using the ELISA described above.

The kinetics for the antibody cell reaction was calculated using thedirect kinetics KinExA software. The OD450 value for each time point wasfitted using the previously calculated Kd and antigen concentration andthe Kon and Koff for the reaction calculated. The results are shown inTable 5.

EXAMPLE 5 ADCC Assays with CD20 Binding Molecules

In this example, AME 5, AME 33, and AME 6F1 IgGs, as well as the C2B8antibody, were tested for their ability to mediate antibody-dependentcell mediated cytotoxicity (ADCC) using human peripheral bloodmononuclear cells as effectors and B lymphoma cell lines as targets.These assays were performed as described below.

PBMC (Peripheral Blood Mononuclear Cell) Isolation

Peripheral blood from healthy donors was diluted 1:2 with phosphatebuffered saline (PBS), pH 7.0. Twelve mL of Histopaque-1077 (Sigma Cat.No. 1077-1) was carefully layered underneath the diluted sample followedby centrifugation in a Sorvall RT6000B centrifuge with swinging bucketrotor at 1000 rpm for 10 minutes with the brake turned off. ThePBMC-containing interphase was collected and washed 3 times with Hanks'Balanced Salt Solution (Gibco). The washed cell pellet was suspended in20 mL RPMI 1640 Media (ATCC) containing 10% Fetal Bovine Serum (FBS)(Omega Scientific). The resuspended PBMCs were split into two T-175culture flasks and 30 mL of RPMI/10% FBS was added to each. Flasks wereincubated overnight in a 37° C./5% CO₂ incubator. The following day thenonadherent PBMCs were collected, centrifuged as above, resuspended inRPMI containing 1% FBS and counted using a hemocytometer.

Target Cell Lines

B lymphoma cell lines were obtained from ATCC and grown as recommended.The day before the experiment the cells were split 1:2. The next day theconcentration was adjusted to 0.5 to 1×10⁶ cells/mL and 50 μL (25,000 to50,000 cells/well) aliquots added to a Becton Dickinson 96-well U-bottomtissue culture plate.

IgG Titrations

IgG dilutions were prepared by diluting the samples in RPMI containing1% FBS. Fifty microliter aliquots of IgG were added to the target cellsin a 96-well microtiter plate and mixed by gentle pipetting. The IgGswere incubated with the target cells for 15 minutes at 37° C./5% CO₂prior to adding the effector cells.

Effector Cells

One hundred microliters of the resuspended PBMCs were added to each wellof the target cell/IgG plate. The concentration of the PBMCs wasadjusted so that the effector:target ratio was in the range of 10-20:1.The plates were incubated at 37° C./5% CO₂ for 3-4 hours.

LDH-Release Detection

Following incubation, the plate was centrifuged at 1-2000 rpm for 5-10min. Fifty μL of the supernatant was carefully removed avoiding pelletedcells. This supernatant was added directly to a Dynex Immulon 2HB flatbottom plate containing 50 μL of PBS per well. To this plate was added100 μl of LDH detection reagent (Roche). The plate was then incubatedfor approximately 15-30 minutes and OD read at 490 nm using a MolecularDevices Vmax Kinetic Microplate Reader.

Data Analysis

Data were plotted as log IgG concentration vs. A490 (See FIG. 15). A490was converted into % cytotoxicity using the following equation: %cytotoxicity=(experimental A490−basal A490)/(maximal A490−basalA490)×100, with maximal A490 determined by adding 2% Triton X-100 to thetarget cells and basal-release measured for a mixture of effector andtarget cells in the absence of sensitizing IgG. Based on the data shownin FIG. 15, it was shown that the EC50 of AME 33 with the Fc mutationsD280H and K290S were consistently 1.5-2.0 times lower than C2B8. Thisdata is also presented in Table 6 below. TABLE 6 Estimated EC50s, ug/ml(Wil-2 cells as target) C2B8 0.0042 +/− .0008 AME 33 0.0063 +/− .0031AME 33 D280H 0.0031 +/− .0009 AME 33 K290S 0.0021 +/− .0005

EXAMPLE 6 Glycoengineering of CD20 Binding Molecules

This example describes glycoengineering methods that were applied tocertain CD20 binding molecules. In particular, AME 1C2 IgG bearingwild-type or mutant (D280H or K290S) Fc regions was coexpressed in CHOcells together with the enzyme β(1-4)-N-acetylglucosaminyltransferaseIII to increase expression of bisected oligosaccharides in the Fcregion. The combination of glycoengineering and mutant Fc regiondecreased the EC50 of 1C2 in ADCC assays relative to unmodified 1C2 orthe commercial RITUXAN antibody. The method was performed as describedbelow.

The β(1-4)-N-acetylglucosaminyltransferase III gene was PCR amplifiedfrom rat kidney cDNA (Clontech, Palo Alto, Calif.). The PCR primersintroduced a NheI site at the 5′-end of the gene and a EcoRI site at the3′-end of the gene (forward primer was5′-GGCGGCTAGCATGAGACGCTACAAGCTTTTTCTCATGTTCTG-3′, SEQ ID NO:103, and thereverse primer was 5′-GGCGGAATTCCTAGCCCTCCGTTGTATCCAACTTGC-3′, SEQ IDNO:104). Following restriction digestion and purification of the PCRproduct it was ligated into similarly cleaved pcDNA3.1 NEO (Invitrogen,Carlsbad, Calif.). The ligated plasmid DNA was used to transform E. coliby electroporation. Ampicillin resistant E. coli colonies were screenedfor the presence of the rat β(1-4)-N-acetylglucosaminyltransferase IIIgene using colony PCR. Plasmid DNA was isolated from positive clones andsequenced. Sequence confirmed plasmid DNA was linearized with BspHI andabout 10 micrograms of the linear DNA used to transfect CHO K1 cellsusing Lipofectamine 2000 (Invitrogen, Carlsbad, Calif.) following themanufacturer's protocol. Following G418 selection, isolated resistantcolonies were expanded in tissue culture. mRNA was isolated and screenedfor the qualitative production of the ratβ(1-4)-N-acetylglucosaminyltransferase III messenger RNA. Each of thesecell lines yielded a PCR product of the anticipated size whereasuntransfected CHOK1 cells did not. DNA encoding anti-CD20 IgG was usedto transfect stable cell lines. Approximately three days followingtransfection, cell culture supernatants were collected and the IgGaffinity purified using protein A chromatography. Following elution ofthe IgGs from the protein A column the samples were dialyzed and theprotein concentration quantitated by measuring its absorbance at 280 nm.

The IgG samples expressed from the different CHOK1 cell lines weretested for the ability to elicit antibody dependent cell-mediatedcytotoxicity (ADCC) using methods described in Example 5. Peripheralblood lymphocytes, isolated from fresh human blood were used as theeffector cells and WIL.2s B cells were used as the target cells in theseexperiments. The amount of target cell lysis was measured using astandard lactate dehydrogenase release assay. The IgG expressed from theengineered CHOK1 cell lines were compared directly with IgG expressedfrom non-engineered CHOK1 cell lines and commercial RITUXAN antibody forthe ability to elicit ADCC. In addition the presence of a bisectingN-acetylglucosamine sugar moiety was confirmed by direct glycosylationanalysis. The results of the ADCC assays for AME 1C2 are shown in FIG.16.

EXAMPLE 7 In Vitro Apoptosis Assays

This example describes certain in vitro apoptosis assays conducted withvarious CD20 binding molecules. In particular, AME 5, AME 33 and AME 6F1(all as full antibodies), as well as the C2B8 antibody, were tested fortheir ability to induce apoptosis of the Ramos B lymphoma cell linefollowing cross-linking with anti-human IgG. These assays were performedas described below.

Annexin V-FITC Staining

The Ramos B lymphoma cell line (ATCC) was split 1:2 24 hours before use.On the day of assay, Ramos cells were centrifuged (1,000 rpm for 5min.), washed with PBS, and resuspended in RPMI plus 10%heat-inactivated FBS (media) at a cell density of 5×10⁵ cells/ml. Onemillion cells were added per well of a 6 well tissue culture plate.Primary antibody was added to the cells at the indicated concentrations,plates were gently shaken for 15 minutes and then returned to 37° C. foran additional 45 minutes. Two ml of pre-warmed goat anti-human IgG (10μg/ml) or medium were added to specified wells and the plates wereincubated at 37° C. for 6 hours.

Cells were harvested by centrifugation (1,000 rpm for 5 min.) andresuspended in 100 μl cold 1× binding buffer (diluted in water from 10×stock) and 10 μl Annexin V-FITC (Southern Biotech). Samples weretransferred to polystyrene round-bottom tubes (Falcon 2058) andincubated in the dark for 15 minutes at room temperature. Binding buffercontaining propidium iodide, 2 μg/ml, was added and samples wereanalyzed on a Becton Dickinson Facscan flow cytometer using CellQuest orWinMDI software for data analysis. Cell death (% apoptotic plus necroticcells) was determined by measuring the percentage of cells staining withAnnexin V. The results of this assay showed that AME 5, 33 and 6F1induced cell death at levels similar to that observed with C2B8 antibodytreatment.

EXAMPLE 8 Complement Mediated Cytotoxicity Assays

This example describes how the C2B8 antibody, Synagis (control) and AMEantibodies 6F1, 2C2, 1D10, 15, 18, and 33 were tested for their abilityto mediate complement-dependent cytotoxicity using human complement andthe Wil-2 B lymphoma cell line as target.

Wil2-S B-lymphoma cells (ATCC) were resuspended at 5×10⁵/ml in RPMI+10%heat-inactivated FBS. Fifty microliters of Wil2-S cell suspension, 50 μlIgG (ranging in concentration from 1 ng/ml to 75 μg/ml), and 50 μldiluted [1:5] human complement were mixed in a 96 well plate (Costar3917). Antibody and complement were diluted in RPMI+10% FBS. Antibodyand complement are diluted in RPMI+10% FBS. The cells were incubated for90 minutes at 37° in 5% CO₂. Alamar Blue, 15 μl per well, was added andthe plate incubated overnight at 37° in 5% CO₂. Fluorescence (reflectingthe number of live cells) was recorded using 560 nm excitation anddetecting emission at 590 nm. The results of these assays show that theEC50s of all AME antibodies tested was similar to that of the C2B8antibody.

EXAMPLE 9 In Vivo CD20 Binding Molecule Administration

This example describes assays that were used to test a number of CD20binding molecules in vivo.

Antibodies AME 45H, 1C2, 5-3, as well as the C2B8 antibody, were assayedin mice as follows. Male 6-8 week-old C.B.17-SCID mice (Taconic) wereinjected s.c. in the right and left flanks with 5×10⁶ Raji cells (ATCC).Antibodies were injected intraperitoneally at 0.01 mg/kg, 0.05 mg/kg,0.1 mg/kg and 0.5 mg/kg approximately 2-3 hrs later (Day 0). Tumorlength, width and height were measured by caliper every Monday,Wednesday and Friday and tumor volume calculated. EC₅₀ values weredetermined from the dose-response curves. The EC50's of AME 4H5, 1C2,and 5-3 were not consistently different than the EC50 for the C2B8antibody.

Antibody AME 33 and the C2B8 antibody were also assayed in mice that hadbeen injected with 5×10⁶ Raji cells. Mice were treated on day three with0.5 mg/kg AME 33 or C2B8. Control mice received saline. The results(shown in FIG. 17) show that AME 33 and C2B8 inhibited the growth ofRaji tumors in a similar manner, with AME 33 allowing less tumor growthover time compared to C2B8.

EXAMPLE 10 Use of CD20 Binding Molecules to Treat Disease in Humans

This example describes the use of CD20 binding molecules for thetherapeutic and prophylactic treatment of certain diseases in a humanpatient including, but not limited to, a disorder selected fromNon-Hodgkin's Lymphoma (NHL) and systemic lupus erythematosus (SLE).

For example, a patient with one of the diseases listed above may beadministered a CD20 binding molecule such as AME 21E1 Hum, AME 6F1, AME2C2, AME 1D10, AME 15, AME 18, AME 33, AME 5-3, AME 1C2, AME 4H5, or AME5, intravenously at 0.4 to 20.0 mg/kg body weight. A typical dosageschedule, may be, for example, 375 mg/m² of the antibody administered asa slow IV infusion once weekly for 4 or 8 doses. Additional dosageregimes are provided in U.S. Pat. No. 6,399,061 to Anderson, hereinincorporated by reference in its entirety for all purposes. Theantibody, or Fab fragment, may also be radiolabeled (e.g. withYttrium-[90]) for therapy and/or in vivo imaging procedures (See, U.S.Pat. No. 6,399,061). Response to therapy may be monitored to determinethe need for increased or reduced dosage and the need for repeattreatment. Additional guidance on response to therapy and dosageschedules is found in U.S. Pat. No. 6,399,061.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inchemistry, medicine, and molecular biology or related fields areintended to be within the scope of the following claims.

1-33. (canceled)
 34. A composition comprising a CD20 binding molecule, wherein the CD20 binding molecule comprises: a) a light chain variable region, wherein the light chain variable region comprises: i) a CDRL1 amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5; ii) a CDRL2 amino acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, and SEQ ID NO:13; iii) a CDRL3 amino acid sequence selected from the group consisting of SEQ ID NO:17, SEQ ID NO:19, and SEQ ID NO:21; iv) an FRL1 amino acid sequence consisting of SEQ ID NO:71; v) an FRL2 amino acid sequence consisting of SEQ ID NO:72; vi) an FRL3 amino acid sequence consisting of SEQ ID NO:73; and vii) an FRL4 amino acid sequence consisting of SEQ ID NO:74. b) a heavy chain variable region, wherein the heavy chain variable region comprises: i) a CDRH1 amino acid sequence selected from the group consisting of SEQ ID NO:23 and SEQ ID NO:25; ii) a CDRH2 amino acid sequence selected from the group consisting of SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, and SEQ ID NO:39; iii) a CDRH3 amino acid sequence selected from the group consisting of SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, and SEQ ID NO:57; iv) an FRH1 amino acid sequence consisting of SEQ ID NO:79; v) an FRH2 amino acid sequence consisting of SEQ ID NO:80; vi) an FRH3 amino acid sequence consisting of SEQ ID NO:81; and vii) an FRH4 amino acid sequence consisting of SEQ ID NO:82.
 35. The composition of claim 34, wherein the CD20 binding molecule comprises the AME 33 Fab.
 36. The composition of claim 34, wherein the CD20 binding molecule has a binding affinity (K_(d)) for human CD20 of 5.0×10⁻¹⁰ M or less, and a dissociation rate (koff) for human CD20 of 5.0×10⁻⁴ s⁻¹ or less.
 37. The composition of claim 36, wherein the CD20 binding molecule has a binding affinity (K_(d)) for human CD20 of 1.5×10⁻¹⁰ M or less.
 38. The composition of claim 36, wherein the CD20 binding molecule has a dissociation rate (k_(off)) for human CD20 of 2.5×10⁻⁴ s⁻¹ or less.
 39. The composition of claim 36, wherein the CD20 binding molecule has an association rate (k_(on)) for human CD20 of 5.0×10⁵ M⁻¹ s⁻¹ or greater.
 40. A method of treating B cell lymphoma comprising administering to a subject a composition comprising a CD20 binding molecule, wherein the CD20 binding molecule comprises: a) a light chain variable region, wherein the light chain variable region comprises: i) a CDRL1 amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5; ii) a CDRL2 amino acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, and SEQ ID NO:13; iii) a CDRL3 amino acid sequence selected from the group consisting of SEQ ID NO:17, SEQ ID NO:19, and SEQ ID NO:21; iv) an FRL1 amino acid sequence consisting of SEQ ID NO:71; v) an FRL2 amino acid sequence consisting of SEQ ID NO:72; vi) an FRL3 amino acid sequence consisting of SEQ ID NO:73; and vii) an FRL4 amino acid sequence consisting of SEQ ID NO:74. b) a heavy chain variable region, wherein the heavy chain variable region comprises: i) a CDRH1 amino acid sequence selected from the group consisting of SEQ ID NO:23 and SEQ ID NO:25; ii) a CDRH2 amino acid sequence selected from the group consisting of SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, and SEQ ID NO:39; iii) a CDRH3 amino acid sequence selected from the group consisting of SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, and SEQ ID NO:57; iv) an FRH1 amino acid sequence consisting of SEQ ID NO:79; v) an FRH2 amino acid sequence consisting of SEQ ID NO:80; vi) an FRH3 amino acid sequence consisting of SEQ ID NO:81; and vii) an FRH4 amino acid sequence consisting of SEQ ID NO:82.
 41. The method of claim 40, wherein the CD20 binding molecule comprises the AME 33 Fab.
 42. The method of claim 40, wherein the CD20 binding molecule has a binding affinity (K_(d)) for human CD20 of 5.0×10⁻¹⁰ M or less, and a dissociation rate (koff) for human CD20 of 5.0×10⁻⁴ s⁻¹ or less.
 43. The method of claim 42, wherein the CD20 binding molecule has a binding affinity (K_(d)) for human CD20 of 1.5×10⁻¹⁰ M or less.
 44. The method of claim 42, wherein the CD20 binding molecule has a dissociation rate (k_(off)) for human CD20 of 2.5×10⁻⁴ s⁻¹ or less.
 45. The method of claim 42, wherein the CD20 binding molecule has an association rate (k_(on)) for human CD20 of 5.0×10⁵ M⁻¹ s⁻¹ or greater.
 46. The method of claim 40, wherein the B cell lymphoma is Non-Hodgkin's lymphoma.
 47. The method of claim 46, wherein the Non-Hodgkin's lymphoma is Waldenstrom's macroglobulinemia. 